Xanthophyll compositions and methods of use

ABSTRACT

The present invention encompasses a carotenoid composition, a process for producing a carotenoid composition, and methods of use thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/106,012, filedDec. 13, 2013 which claims priority to U.S. Ser. No. 61/739,074, filedDec. 19, 2012, each of which is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention encompasses a carotenoid composition, a processfor producing a carotenoid composition, and methods of use thereof.

BACKGROUND OF THE INVENTION

Carotenoids, including lutein and other xanthophylls, are naturalcompounds used in pigmenting compositions. They may be found in extractsof several different plants, including marigold and paprika plants.These plant extracts, referred to as oleoresins, are processed intoformulations of carotenoids that may be used in a variety of industries.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a schematic of a continuous flow apparatus describedherein.

FIGS. 2A-B show the results of polarizing microscopy at 100magnifications. FIG. 2A shows X-40; FIG. 2B shows XCT.

FIGS. 3A-B show the results of optical microscopy at 400 magnifications.FIG. 3A shows X-40; FIG. 3B shows XCT.

FIG. 4 shows the DSC curves of XCT and X-40.

FIG. 5A-D shows melting of X-40. FIG. 5A shows melting at 40° C.; FIG.5B shows melting at 80° C.; FIG. 5C shows melting at 150° C.; and FIG.5D shows melting at 200° C.

FIG. 6. provides a schematic for a comparitive X-40 process.

FIGS. 7A-B shows comparitive stability studies between XCT, X-40,Competitor Product 3, Competitor Product 4, Competitor Product 5, andCompetitor Product 6.

FIGS. 8A-D show the results of Minolta color analysis and DSM/Rochecolor fan for studies where no red pigment is added against alternativeproducts.

FIGS. 9A-B show the total xanthophylls in egg when XCT is administeredagainst alternative products.

FIGS. 10A-D show the results of Minolta color analysis and DSM/Rochecolor fan for studies where no red pigment is added against alternativeproducts.

FIGS. 11A-B show the total xanthophylls in egg when XCT is administeredagainst alternative products.

FIG. 12A-D show the total xanthophylls in egg when XCT is administeredagainst apo-ester (β-apo-8′-carotenoic acid ethyl ester).

FIG. 13 shows DSM/Roche color fan results when synthetic red dye isadded.

FIG. 14 shows DSM/Roche color fan results when natural red dye is added.

FIGS. 15A-C show the results of various spectroscopic studies.

FIG. 15A shows the FTIR spectra for X-40 and XCT. FIG. 15B shows anexpansion of the region from 1800 to 600 cm⁻¹. FIG. 15C shows Ramanspectra of X-40 and XCT.

FIGS. 16A-C show the results of Thin Layer Chromatography (TLC) of X-40and XCT.

SUMMARY OF THE INVENTION

In some aspects, the present disclosure provides a composition, thecomposition comprising soap derived from the saponification of a naturalcarotenoid-containing oleoresin, wherein the soap containsnon-esterified xanthophyll particles, and retains greater than 80% totalxanthophyll concentration when stored at room temperature, in an oxygenpermeable dark bag, for three months.

In another aspect, the present disclosure provides a compositioncomprising soap derived from the saponification of a naturalcarotenoid-containing oleoresin, wherein the soap containsnon-esterified xanthophyll particles, and 90% of the non-esterifiedxanthophyll particles are less than 0.5 microns across the largestdiameter.

In still another aspect, a method for creating a final product with anon-esterified carotenoid composition of greater than 10% is provided.The process comprises (a) alkaline saponification of a naturalcarotenoid-containing oleoresin, wherein the saponification occurs inthe presence of a metal hydroxide, with intimate mixing, and occurs at atemperature between about 110° C. to about 180° C., resulting in acomposition comprising non-esterified carotenoids, (b) atomization ofthe resulting soap comprising non-esterified carotenoids to produce anatomized soap, and (c) isomerization of the non-esterified carotenoids,wherein the atomized soap is heated such that greater than 80% of thenon-esterified carotenoids present are in the all-trans isomerconfiguration, and the non-esterified carotenoid concentration of thefinal soap product is greater than 10%.

In still another alternative aspect, a process for creating an aqueousproduct from water and a final product with a non-esterified carotenoidconcentration of greater than 10% is provided. The process comprises (a)alkaline saponification of a natural carotenoid-containing oleoresin,wherein the saponification occurs in the presence of a metal hydroxide,with intimate mixing, and occurs at a temperature between about 110° C.to about 180° C., resulting in a soap comprising non-esterifiedcarotenoids, (b) isomerization of the non-esterified carotenoids,wherein the soap is heated such that greater than 80% of thenon-esterified carotenoids present are in the all-trans isomerconfiguration, to produce a non-esterified carotenoid concentration ofthe final soap product is greater than 10%, and (c) contacting the finalsoap product with water in sufficient amounts to create an aqueousproduct having from 5% to about 25% of the final soap product.

Other aspects and features of the present disclosure are providedherein.

DETAILED DESCRIPTION OF THE INVENTION

The present invention encompasses a carotenoid composition, a process ofproducing the carotenoid formulation, and methods of using thecarotenoid composition. Advantageously, a carotenoid composition of theinvention has improved bioavailability and increased coloring efficiencycompared to carotenoid compositions produced by other means.

I. Composition

One aspect of the present invention encompasses a composition comprisinga soap derived from the saponification of a naturalcarotenoid-containing oleoresin. Such a soap contains non-esterifiedxanthophyll particles. A soap of the invention also comprises theremaining components of the original natural carotenoid-containingoleoresin.

A composition of the invention may either be liquid or a solid, e.g.granules or a powder. Generally speaking the moisture content of a solidcomposition of the invention is below 10%. In some embodiments, themoisture content is below 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%. Inexemplary embodiments, the moisture content is below 3%. In furtherexemplary embodiments, the moisture content is below 2%.

(a) Natural Carotenoid-Containing Oleoresin

A composition of the invention comprises a soap derived from thesaponification of a natural carotenoid-containing oleoresin. The term“oleoresin,” as used herein, refers to a composition comprising a plantextract. The phrase “carotenoid-containing oleoresin” refers to anoleoresin that contains one or more carotenoids, which are organicpigments. Finally, as used herein, the phrase “naturalcarotenoid-containing oleoresin” refers to a carotenoid-containingoleoresin derived from a plant.

Suitable examples of natural carotenoid-containing oleoresins are knownin the art. For instance, a natural carotenoid-containing oleoresin maybe a marigold oleoresin, or may be a paprika oleoresin, or a combinationthereof. In one embodiment the natural carotenoid-containing oleoresinis a marigold oleoresin. For instance, the marigold oleoresin may be aTagetes erecta, Tagetes patula, Tagetes tenuifolia, Tagetes pumila or aTagetes hybrid oleoresin. In another embodiment, the naturalcarotenoid-containing oleoresin is a paprika oleoresin. For instance,the paprika oleoresin may be a Capsicum annumlinn, Capsicum bacatum,Capsicum buforum or Capsicum frutescens oleoresin. In other embodiments,the natural carotenoid-containing oleoresin is a combination of marigoldand paprika oleoresin. For instance, a natural carotenoid-containingoleoresin may comprise a ratio of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1,4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10marigold oleoresin to paprika oleoresin.

Methods of preparing an oleoresin from a plant are known in the art.Alternatively, the oleoresin may be purchased.

(b) Non-Esterified Xanthophyll Particles

A composition of the invention comprises non-esterified xanthophyllparticles. As used herein, the phrase “xanthophyll particles” refers toxanthophyll, regardless of whether the xanthophyll is crystalline oramorphous. Various ratios of crystalline xanthophyll to amorphousxanthophyll are envisioned, ranging from about 100% crystalline to about100% amorphous. In one embodiment, a composition of the inventioncomprises both crystalline and amorphous xanthophyll. In anotherembodiment, a composition of the invention comprises at least about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% crystalline xanthophyll. In yetanother embodiment, a composition of the invention comprises at leastabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% amorphous xanthophyll.

The phrase “non-esterified” as used herein, refers to xanthophyll thathas been hydrolyzed from a fatty acid ester. As detailed in Section IIIof this application, a xanthophyll may be hydrolyzed from a fatty acidester via saponification of the natural carotenoid-containing oleoresin.

The term “xanthophyll” refers to a carotenoid that comprises at leastone oxygen atom. Such a compound may also be referred to as aphylloxanthin. Suitable xanthophylls include those found in a naturalcarotenoid-containing oleoresin. For example, suitable xanthophylls mayinclude lutein, zeaxanthin, neoxanthin, violaxanthin, α- andβ-cryptoxanthin, capsanthin and capsorubin. In one embodiment, acomposition of the invention comprises at least one type of xanthophyll.In another embodiment, a composition of the invention comprises at leasttwo types of xanthophylls. In yet another embodiment, a composition ofthe invention comprises at least three types of xanthophylls. In anexemplary embodiment, a composition of the invention comprises lutein.In another exemplary embodiment, a composition of the inventioncomprises zeaxanthin. In a further exemplary embodiment, a compositionof the invention comprises both lutein and zeaxanthin.

i. Size Across the Largest Diameter

Generally speaking, the size of the xanthophyll particles in acomposition of the invention is small, e.g. less than 1 micron. Thisrefers to the size of the largest diameter of the particle. This smallsize contributes to both increased bioavailability and increasedstability, when compared to larger particle sizes.

In some embodiments, at least about 90, 91, 92, 93, 94, 95, 96, 97, 98,99, or 100% of the non-esterified xanthophyll particles are less than1.0 microns across the largest diameter. In other embodiments, at leastabout 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of thenon-esterified xanthophyll particles are less than 0.9 microns acrossthe largest diameter. In certain embodiments, at least about 90, 91, 92,93, 94, 95, 96, 97, 98, 99, or 100% of the non-esterified xanthophyllparticles are less than 0.8 microns across the largest diameter. Inseveral embodiments, at least about 90, 91, 92, 93, 94, 95, 96, 97, 98,99, or 100% of the non-esterified xanthophyll particles are less than0.7 microns across the largest diameter. In select embodiments, at leastabout 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100% of the non-esterified xanthophyll particles areless than 0.6 microns across the largest diameter.

In one embodiment, at least about 75% of the non-esterified xanthophyllparticles are less than 0.5 microns across the largest diameter. Forinstance, in some embodiments, at least about 75, 76, 77, 78, 79, 80,81, 82, 83, 84, 85, 86, 87, 88, 89, or 90% of the non-esterifiedxanthophyll particles are less than 0.5 microns across the largestdiameter. In other embodiments, at least about 91, 92, 93, 94, 95, 96,97, 98, or 99% of the non-esterified xanthophyll particles are less than0.5 microns across the largest diameter. In one embodiment, at leastabout 90% of the non-esterified xanthophyll particles are less than 0.5microns across the largest diameter.

In an alternative embodiment, at least about 75% of the non-esterifiedxanthophyll particles are less than 0.4 microns across the largestdiameter. For instance, in some embodiments, at least about 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or 90% of thenon-esterified xanthophyll particles are less than 0.4 microns acrossthe largest diameter.

ii. Amount of Non-Esterified Xanthophyll Particles

Advantageously, a composition of the invention comprises a highconcentration of non-esterified xanthophyll particles. For instance, inone embodiment, a composition of the invention comprises about 75 mg ofnon-esterified xanthophyll per gram of soap derived from thesaponification of a natural carotenoid-containing oleoresin. In otherembodiments, a composition of the invention comprises at least about 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, or110 mg of non-esterified xanthophyll per gram of soap derived from thesaponification of a natural carotenoid-containing oleoresin. In stillother embodiments, a composition of the invention comprises about 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119 or 120 mg of non-esterified xanthophyll per gramof soap derived from the saponification of a naturalcarotenoid-containing oleoresin. In an exemplary embodiment, acomposition of the invention comprises about 90 mg to about 110 mg ofnon-esterified xanthophyll per gram of soap derived from thesaponification of a natural carotenoid-containing oleoresin. In afurther exemplary embodiment, a composition of the invention comprisesat least about 99 mg of non-esterified xanthophyll per gram of soapderived from the saponification of a natural carotenoid-containingoleoresin. In an embodiment where dioxin removal from the oleoresin isconducted, a composition of the invention comprises about 110, 111, 112,113, 114, 115, 116, 117, 118, 119 or 120, 121, 122, 123, 124, 125, 126,127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140 mgof non-esterified xanthophyll per gram of soap derived from thesaponification of a natural carotenoid-containing oleoresin.

In certain embodiments, a dry composition of the invention is at least5, at least 6, at least 7, at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15% xanthophyll orhigher. For instance, a dry composition of the invention may be at leastabout 8, 8.25, 8.5, 8.75, 9, 9.25, 9.5, 9.75, 10, 10.25, 10.5, 10.75, or11% xanthophyll. In an exemplary embodiment, a dry composition of theinvention is at least about 9.5% xanthophyll. In a further exemplaryembodiment, a dry composition of the invention is at least about 10%xanthophyll. In another alternative embodiment, with dioxin removal fromthe oleoresin, the concentration of xanthophyll can go higher. In suchaspects, the composition may be at least 9, 9.25, 9.5, 9.75, 10, 10.25,10.5, 10.75, 11, 11.25, 11.5, 11.75, 12, 12.25, 12.5, 12.75, 13, 13.25,13.5, 13.75, 14, 14.25, 14.5, 14.75, 15, 15.25, 15.5, 15.75, 16% orhigher.

In other embodiments, a liquid composition of the invention is at least0.5, at least 1, at least 2, or at least 3% xanthophyll. For instance, aliquid composition of the invention may be at least about 0.5, 0.75,1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, or 3.0% xanthophyll. In anexemplary embodiment, a liquid composition of the invention is at leastabout 1% xanthophyll. In a further exemplary embodiment, a liquidcomposition of the invention comprises between about 1% and about 3%xanthophyll.

iii. Isomerism

As is known in the art, xanthophylls may exist as different isomers. Inparticular, they may exist as cis isomers, or all trans isomers. Methodsof determining the amount of a cis isomer, or the amount of all transisomer, in a composition are also known in the art. For instance, seethe Examples included herewith. Generally speaking, a composition of theinvention may comprise at least about 50% of the all trans isomer. Insome embodiments, a composition of the invention may comprise at leastabout 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99% of the all transisomer. In other embodiments, a composition of the invention maycomprise at least about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or 85%of the all trans isomer. In exemplary embodiments, a composition of theinvention may comprise at least about 80% of the all trans isomer.

(c) Stability

The xanthophyll particles of a composition of the invention areremarkably stable. For instance, if a composition of the invention isstored at room temperature, in an oxygen permeable bag, at least about80% of the initial xanthophyll concentration is present at one month. Inone embodiment, at room temperature and in an oxygen permeable bag, atleast about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, or 100% of the initial total xanthophyllconcentration is present at one month. In an exemplary embodiment, atroom temperature and in an oxygen permeable bag, at least about 98, 99,or 100% of the initial total xanthophyll concentration is present at onemonth.

In another embodiment, at room temperature and in an oxygen permeablebag, at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, or 100% of the initial total xanthophyllconcentration is present at three months. In an exemplary embodiment, atroom temperature and in an oxygen permeable bag, at least about 92, 93,94, 95, 96, 97, 98, 99, or 100% of the initial total xanthophyllconcentration is present at three months.

In still another embodiment, at room temperature and in an oxygenpermeable bag, at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, or 98% of the initial total xanthophyll concentration ispresent at six months. In an exemplary embodiment, at room temperatureand in an oxygen permeable bag, at least about 90% of the initial totalxanthophyll concentration is present at six months.

In yet another embodiment, at room temperature and in an oxygenpermeable bag, at least about 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, or 98% of the initial total xanthophyll concentration ispresent at nine months. In an exemplary embodiment, at room temperatureand in an oxygen permeable bag, at least about 85, 86, 87, 88, 89, or90% of the initial total xanthophyll concentration is present at ninemonths.

In embodiments where a composition of the invention is stored at 50° C.in an impermeable bag, at least about 90, 91, 92, 93, 94, 95, 96, 97,98, 99, or 100% of the initial total xanthophyll concentration ispresent at up to about six months. In an exemplary embodiment, at leastabout 95, 96, 97, 98, 99, or 100% of the initial total xanthophyllconcentration is present at up to about six months.

In other embodiments where a composition of the invention is stored at50° C. in an impermeable bag, at least about 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, or 100% of the initial total xanthophyllconcentration is present at nine months. In an exemplary embodiment, atleast about 85, 86, 87, 88, 89, or 90% of the initial total xanthophyllconcentration is present at nine months.

In a further exemplary embodiment, a composition of the invention, mixedwith animal feed and stored at room temperature in a dark, oxygenpermeable bag, maintains at least about 90% of the initial xanthophyllconcentration after one month, and in some embodiments, maintains atleast 90, 91, 92, 93, or 94% of the initial xanthophyll concentrationafter one month. In a still further exemplary embodiment, a compositionof the invention, mixed with animal feed and stored at room temperaturein a dark, oxygen permeable bag, maintains at least about 80% of theinitial xanthophyll concentration after three, four, or six months, andin some embodiments, maintains at least about 80, 81, 82, 83, 84, 85,86, 87, 88, 89, or 90% of the initial xanthophyll concentration afterthree, four, or six months.

(d) Formulations

A composition of the invention may either be a liquid or a solid. Whenthe composition is a solid, the soap particles (which are comprised, inpart, of non-esterified xanthophyll particles) are typically betweenabout 40 microns to about 300 microns across the longest particlediameter. These particles may also form aggregates, ranging in size upto about 850 microns. In one embodiment, 90% of the soap particles oraggregates are between about 40 microns and about 850 microns. Inanother embodiment, 90% of the soap particles or aggregates are betweenabout 70 microns and 700 microns. In yet another embodiment, 90% of thesoap particles or aggregates are between about 100 microns and 550microns. In still yet another embodiment, 90% of the soap particles oraggregates are between about 200 microns and about 500 microns. Such asoap contains non-esterified xanthophyll particles.

In certain embodiments, a composition of the invention may be formulatedby itself, or as a part of a feed. A dry feed supplement may beuniformly dispersed throughout a liquid, a liquid food, a dry food,grain, protein products, feed supplements, or mixtures thereof.

In some embodiments, a composition of the invention may be formulated asan aqueous formulation. An aqueous formulation may be a solution,dispersion, or an emulsion. The aqueous formulation may be addeddirectly to the drinking water of an animal or it may be mixed into orapplied to a dry or liquid food.

(e) Antioxidants

A composition of the invention may also include at least oneantioxidant. A variety of antioxidants or combination of antioxidantsare suitable for use in a composition of the invention. The antioxidantmay comprise a quinoline compound. Typically, the quinoline compoundwill be a substituted 1,2-dihydroquinoline. Substituted1,2-dihydroquinoline compounds suitable for use in the inventiongenerally comprise Formula (I):

wherein:

R¹, R², R³ and R⁴ are independently selected from the group consistingof hydrogen and an alkyl group having from 1 to about 6 carbons; and

R⁵ is an alkoxy group having from 1 to about 12 carbons.

In an iteration, the substituted 1,2-dihydroquinoline comprises Formula(I), wherein:

R¹, R², R³ and R⁴ are independently selected from the group consistingof hydrogen and an alkyl group having from 1 to about 4 carbons;

and R⁵ is an alkoxy group having from 1 to about 4 carbons.

In one preferred embodiment, the substituted 1,2-dihydroquinoline willbe 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline comprising Formula(II):

The compound, 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline, commonlyknown as ethoxyquin, is sold under the trademark AGRADO®. The presentinvention also encompasses salts of ethoxyquin and other compoundscomprising Formula (I). Ethoxyquin and other compounds having Formula(I) may be purchased commercially from Novus International, Inc. (St.Louis, Mo.) or made in accordance with methods generally known in theart, for example, as detailed in U.S. Pat. No. 4,772,710, which ishereby incorporated by reference in its entirety.

A variety of other antioxidants are suitable for use in a composition ofthe present invention. In some embodiments, the antioxidant may be acompound that interrupts the free-radical chain of oxidative reactionsby protonating free radicals, thereby inactivating them. Alternatively,the antioxidant may be a compound that scavenges the reactive oxygenspecies. In still other embodiments, the antioxidant may be a syntheticcompound, a semi-synthetic compound, or a natural (or naturally-derived)compound.

Suitable antioxidants may include, but are not limited to, ascorbic acidand its salts, ascorbyl palmitate, ascorbyl stearate, anoxomer,n-acetylcysteine, benzyl isothiocyanate, m-aminobenzoic acid,o-aminobenzoic acid, p-aminobenzoic acid (PABA), butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), caffeic acid,canthaxantin, alpha-carotene, beta-carotene, beta-apo-carotenoic acid,carnosol, carvacrol, catechins, cetyl gallate, chlorogenic acid, citricacid and its salts, clove extract, coffee bean extract, p-coumaric acid,3,4-dihydroxybenzoic acid, N,N′-diphenyl-p-phenylenediamine (DPPD),dilauryl thiodipropionate, distearyl thiodipropionate,2,6-di-tert-butylphenol, dodecyl gallate, edetic acid, ellagic acid,erythorbic acid, sodium erythorbate, esculetin, esculin,6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline (ethoxyquin), ethylgallate, ethyl maltol, ethylenediaminetetraacetic acid (EDTA),eucalyptus extract, eugenol, ferulic acid, flavonoids (e.g., catechin,epicatechin, epicatechin gallate, epigallocatechin (EGC),epigallocatechin gallate (EGCG), polyphenol epigallocatechin-3-gallate,flavones (e.g., apigenin, chrysin, luteolin), flavonols (e.g.,datiscetin, myricetin, daemfero), flavanones, fraxetin, fumaric acid,gallic acid, gentian extract, gluconic acid, glycine, gum guaiacum,hesperetin, alpha-hydroxybenzyl phosphinic acid, hydroxycinammic acid,hydroxyglutaric acid, hydroquinone, n-hydroxysuccinic acid,hydroxytryrosol, hydroxyurea, rice bran extract, lactic acid and itssalts, lecithin, lecithin citrate; r-alpha-lipoic acid, lutein,lycopene, malic acid, maltol, 5-methoxy tryptamine, methyl gallate,monoglyceride citrate; monoisopropyl citrate; morin,beta-naphthoflavone, nordihydroguaiaretic acid (NDGA), octyl gallate,oxalic acid, palmityl citrate, phenothiazine, phosphatidylcholine,phosphoric acid, phosphates, phytic acid, phytylubichromel, pimentoextract, propyl gallate, polyphosphates, quercetin, trans-resveratrol,rosemary extract, rosmarinic acid, sage extract, sesamol, silymarin,sinapic acid, succinic acid, stearyl citrate, syringic acid, tartaricacid, thymol, tocopherols (i.e., alpha-, beta-, gamma- anddelta-tocopherol), tocotrienols (i.e., alpha-, beta-, gamma- anddelta-tocotrienols), tyrosol, vanilic acid,2,6-di-tert-butyl-4-hydroxymethylphenol (i.e., Ionox 100),2,4-(tris-3′,5′-bi-tert-butyl-4′-hydroxybenzyl)-mesitylene (i.e., Ionox330), 2,4,5-trihydroxybutyrophenone, ubiquinone, tertiary butylhydroquinone (TBHQ), thiodipropionic acid, trihydroxy butyrophenone,tryptamine, tyramine, uric acid, vitamin K and derivates, vitamin Q10,wheat germ oil, zeaxanthin, or combinations thereof.

Exemplary antioxidants may include synthetic substituted phenoliccompounds, such as tertiary butyl hydroquinone (TBHQ), butylatedhydroxyanisole (BHA), or butylated hydroxytoluene (BHT);6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline (ethoxyquin); gallic acidderivatives, such as n-propyl gallate; vitamin C derivatives, such asascorbyl palmitate; lecithin; and vitamin E compounds, such as,alpha-tocopherol.

A composition of the invention may comprise at least one antioxidant. Insome embodiments, a composition of the invention may comprise more thanone antioxidant. By formulating a combination of antioxidants in thismanner, a broad spectrum of fat sources, including fat sourcesrelatively high in unsaturated fatty acids, may be utilized with acomposition of the invention, such as in the animal feed ration or watersource.

(f) Other Components

In certain embodiments, a composition of the invention may compriseother components, such as a carrier, a preservative, a free flow agent,etc. In certain embodiments, a composition of the invention may comprisea free flow agent. Suitable free flow agents are known in the art, andmay comprise, for instance, a salt of stearic acid, SiO₂ and/or talc. Insome embodiments, the free flow agent may be present in an amount of 0%to 15% by weight of the total composition, more preferably, the amountof the free flow agent is between 3% and 15%, or between 5% and 10%.Similarly, a composition of the invention may comprise a preservative.Suitable preservatives are known in the art. In still other embodiments,the composition may comprise an emulsifier. Suitable emulsifiers maycomprise, for instance, a non-ionic emulsifier derived from propyleneglycol or polyethyleneglycol ricinoleate (E-484).

(g) Exemplary Embodiments

In an exemplary embodiment, a composition of the invention is derivedfrom marigold oleoresin. In another exemplary embodiment, 90% of thexanthophyll particles of a composition of the invention are less than0.5 microns, the final soap product contains at least about 75 mgxanthophylls per gram of soap, and 80% of the non-esterifiedxanthophylls are the all-trans isomer. In a further exemplaryembodiment, a composition of the invention comprises ethoxyquin. Instill another exemplary embodiment, 98% of the xanthophyll presentinitially remains after one month at room temperature when stored in anoxygen permeable bag.

In another exemplary embodiment, a powder composition of the inventioncomprises between about 70% to about 99% marigold soap, between about 0%to about 15% free flow agent, between about 0.1% to about 3% metalhydroxide, between about 0.1% to about 2% moisture, and about 0% toabout 1% antioxidant, such as ethoxyquin.

In yet another exemplary embodiment, a powder composition of theinvention comprises between about 75% to about 95% marigold soap,between about 0% to about 15% free flow agent, and between about 0% toabout 8% stearic acid.

In still another exemplary embodiment, a powder composition of theinvention comprises between about 75% to about 95% marigold soap,between about 0% to about 15% SiO₂, and between about 0% to about 8%stearic acid.

In a further exemplary embodiment, a powder composition of the inventioncomprises between about 75% to about 95% marigold soap, between about 0%to about 15% talc, and between about 0% to about 8% stearic acid.

In a further exemplary embodiment, a powder composition of the inventioncomprises between about 75% to about 95% marigold soap, between about 0%to about 6% SiO₂, between about 2% to about 10% talc, and between about0% to about 8% stearic acid.

In some exemplary embodiments, a liquid composition of the inventioncomprises between about 5 to about 25% marigold soap, between about 0 toabout 0.6% antioxidant, such as ethoxyquin, between about 0 to about 1%emulsifier, and between about 82 to about 89% water. In a furtherexemplary embodiment, a liquid composition of the invention comprisesbetween 0.5 and 3% xanthophylls, between about 0 to about 0.6%antioxidant, such as ethoxyquin, between about 0 to about 1% emulsifier,and between about 82 to about 89% water.

In other exemplary embodiments, a liquid composition of the inventioncomprises between about 5 to about 25% marigold soap, between about 0 toabout 0.6% antioxidant, such as ethoxyquin, between about 0 to about 1%glyceryl polyethyleneglycol ricinoleate (E-484), and between about 82 toabout 89% water. In a further exemplary embodiment, a liquid compositionof the invention comprises between about 0 to about 3% xanthophylls,between about 0 to about 0.6% antioxidant, such as ethoxyquin, betweenabout 0 to about 1% glyceryl polyethyleneglycol ricinoleate (E-484), andbetween about 82 to about 89% water.

II. Feed Rations

Another aspect of the present invention encompasses an animal feed. Ananimal feed of the invention comprises a composition as detailed inSection I above. The exact formulation of the animal feed composition isnot critical to the present invention. Feed ingredients are selectedaccording to the nutrient requirements of the particular animal forwhich the feed is intended; these requirements depend, interalia, uponthe age and stage of development of the animal, the sex of the animal,and other factors. Feed ingredients may be grouped into eight classes onthe basis of their composition and their use in formulating diets: dryforages and roughages; pasture, range plants and forages fed fresh;silages; energy feeds; protein supplements; mineral supplements; vitaminsupplements; and additives. See National Research Council (U.S.)Subcommittee on Feed Composition, United States-Canadian Tables of FeedComposition, 3d rev., National Academy Press, pp. 2, 145 (1982). Theseclasses are, to a certain extent, arbitrary, as some feed ingredientscould be classified in more than one class. Typically, a feedformulation will also depend upon the costs associated with eachingredient, with the least-expensive composition of ingredients thatgives the needed nutrients being the preferred formulation.

By way of non-limiting example, in one embodiment, the animal feedration is formulated for poultry. As noted above, feed formulationsdepend in part upon the age and stage of development of the animal to befed. Leeson and Summers (Nutrition of the Chicken, 4^(th) ed., pp.502-510, University Books, 2001) describe several representative poultrydiets for pullets, layers, broilers and broiler breeders. For example,most chicken diets contain energy concentrates such as corn, oats,wheat, barley, or sorghum; protein sources such as soybean meal, otheroilseed meals (e.g., peanut, sesame, safflower, sunflower, etc.),cottonseed meal, animal protein sources (meat and bone meal, dried whey,fish meal, etc.), grain legumes (e.g., dry beans, field peas, etc.), andalfalfa; and vitamin and mineral supplements, if necessary (forinstance, meat and bone meal is high in calcium and phosphorous, andthus these minerals do not need to be supplemented in a feed rationcontaining meat and bone meal).

In another embodiment, the animal feed ration is formulated for swine.The feed formulation will vary for piglets, grower and finisher pigs,gilt development, gestating sows, and lactating sows. Swine feedformulations typically comprise grains (e.g., corn, barley, grainsorghum, oats, soybeans, wheat, etc.), crude proteins (e.g., fish meal,gluten meal, meat meal, soybean meal, tankage, which is the residue thatremains after rendering fat in a slaughterhouse, etc.), crude fat (e.g.,fish oils, vegetable oils, animal fats, yellow grease, etc.),supplemental amino acids (e.g., lysine, methionine or methionineanalogs, etc), vitamins, minerals, and other supplemental agents.

In another embodiment, the animal ration is formulated for aquaticanimals, for example in an aquaculture feed. As appreciated by a skilledaquaculturist, the feed formulation depends upon the organism beingcultured and the developmental stage of the organism. Typicalaquaculture preparations contain energy sources, e.g., protein fromanimal blood meal, meat and bone meal, poultry meal, crab meal, fishmeal, shrimp meal, squid meal, and krill; protein/carbohydrates fromplants (e.g., alginates, canola, corn, corn gluten, cottonseed meal,kelp meal, molasses, legumes, peanut meal, rice, soybeans, soy proteinconcentrate, soybean meal, wheat, and wheat gluten); and oils (e.g.,fish oil, vegetable oil). The feed preparation may be furthersupplemented with amino acids (e.g., arginine, histidine, isoleucine,lysine, methionine, phenylalanine, threonine, tryptophan, and valine);vitamins, minerals, and other supplemental agents.

In another embodiment, the animal ration is formulated for a ruminantanimal. The nutrient and energy content of many common ruminant feedingredients have been measured and are available to the public. TheNational Research Council has published books that contain tables ofcommon ruminant feed ingredients and their respective measured nutrientand energy content. Additionally, estimates of nutrient and maintenanceenergy requirements are provided for growing and finishing cattleaccording to the weight of the cattle. National Academy of Sciences,Nutrient Requirements of Beef Cattle, Appendix Tables 1-19, 192-214,National Academy Press, (2000); Nutrient Requirements of Dairy Cattle(2001), each incorporated herein in its entirety. This information canbe utilized by one skilled in the art to estimate the nutritional andmaintenance energy requirements of cattle with non-functional rumens,such as calves under about 500 lbs in weight, or cattle with functionalrumens, such as growing cattle or dairy cattle.

(a) Additional Ingredients

A composition of the invention may be provided to the animal in the formof a feed premix or feed supplement. The premix will generally be addedto various formulations of grain concentrates and forages to formulate atotal animal feed ration. As will be appreciated by the skilled artisan,the particular premix formulation can and will vary depending upon thefeed ration and animal that the feed ration will be fed to. In additionto combinations of the invention, the premix may further optionallyinclude one or more of a mixture of natural amino acids, analogs ofnatural amino acids, vitamins and derivatives thereof, enzymes, animaldrugs, hormones, effective microorganisms, preservatives, and flavors.

In one embodiment, the feed premix may include one or more amino acids.Suitable examples of amino acids, depending upon the formulation, mayinclude alanine, arginine, asparagines, aspartate, cysteine, glutamate,glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline. Other amino acids usable as feed additives include, by way ofnon-limiting example, N-acylamino acids, hydroxy homologue compounds,and physiologically acceptable salts thereof, such as hydrochlorides,hydrosulfates, ammonium salts, potassium salts, calcium salts, magnesiumsalts and sodium salts of amino acids.

In still another embodiment, a feed premix may include vitamins orderivatives of vitamins. Examples of suitable vitamins and derivativesthereof may include vitamin A, vitamin A palmitate, vitamin A acetate,β-carotene, vitamin D (e.g., D₂, D₃, and D₄), vitamin E, menadionesodium bisulfite, vitamin B (e.g., thiamin, thiamin hydrochloride,riboflavin, nicotinic acid, nicotinic amide, calcium pantothenate,pantothenate choline, pyridoxine hydrochloride, cyanocobalamin, biotin,folic acid, p-aminobenzoic acid), vitamin K, vitamin Q, vitamin F, andvitamin C.

In yet another embodiment, a feed premix may include one or moreenzymes. Suitable examples of enzymes may include protease, amylase,lipase, cellulase, xylanase, glucanase, pectinase, phytase,hemicellulase and other physiologically effective enzymes.

In still another embodiment, a feed premix may include a drug approvedfor use in animals. Non-limiting examples of suitable animal drugs mayinclude antibiotics such as tetracycline type (e.g., chlortetracyclineand oxytetracycline), amino sugar type, ionophores (e.g., rumensin,virginiamycin, and bambermycin) and macrolide type antibiotics.

In an additional embodiment, a feed premix may include a hormone.Suitable hormones may include estrogen, stilbestrol, hexestrol,tyroprotein, glucocorticoids, insulin, glucagon, gastrin, calcitonin,somatotropin, and goitradien.

In an additional embodiment, a feed premix may include a substance toincrease the palatability of the feed ration. Suitable examples of suchsubstances may include natural sweeteners, such as molasses, andartificial sweeteners such as saccharin and aspartame.

As will be appreciated by the skilled artisan, any of the substancesthat may be included in a premix comprising a composition of theinvention can be used alone or in combination with one another. Theconcentration of these additives will depend upon the animal the premixis intended for, and the desired result.

(b) Encapsulation

In yet another embodiment, the product is encapsulated. As used herein,an encapsulated composition is a composition that has been packaged in amaterial. In many cases, encapsulation provides protection fromdegradation from light, heat, oxygen and moisture. Encapsulation mayalso provide release under specific conditions or protect an activeingredient from degrading before reaching a point in the digestivesystem where it can be absorbed. Encapsulation, in some instances, isprovided by spray coating or spray drying, extrusion, coating whichgenerally involves forcing a core material in a molten wall materialthrough a die into a bath of desiccant liquid. Upon contacting theliquid, the coating material hardens forming the outer encapsulation.Encapsulation may be provided, in some instances, by inclusioncomplexation, or molecular inclusion, using, for example, cyclodextrin.In still another embodiments, encapsulation may be provided bycoacervation, emulsion phase separation, or liposome entrapment. Inanother, exemplary embodiment, the product is not encapsulated.

III. Process

In another aspect, the invention encompasses a continuous process forcreating a final soap product with a non-esterified carotenoidconcentration of greater than 10%. The process comprises (a) alkalinesaponification of a natural carotenoid-containing oleoresin, wherein thesaponification occurs in the presence of a metal hydroxide, withintimate mixing, and occurs at a temperature between about 110° C. toabout 180° C. resulting in a soap comprising non-esterified carotenoids,(b) atomization of the soap from step (a), and (c) isomerization of thenon-esterified carotenoids, wherein the atomized soap is heated suchthat greater than 80% or more of the non-esterified carotenoids presentin the soap in the all-trans isomer configuration, and thenon-esterified carotenoid concentration of the final product is greaterthan 10%. In certain embodiments, the soap is also dried. Drying may beperformed, for instance, during atomization, after atomization, beforeisomerization, during isomerization, after isomerization, or acombination thereof.

(a) Alkaline Saponification

The process comprises alkaline saponification of a naturalcarotenoid-containing oleoresin, wherein the saponification occurs inthe presence of a metal hydroxide, with intimate mixing, and occurs at atemperature between about 110° C. to about 180° C. resulting in a soapcomposition comprising non-esterified carotenoids. Through this process,the carotenoids found in the natural carotenoid-containing oleoresinwhich are initially bound to fatty acids through ester moieties may bereleased by hydrolysis, or de-esterification from the fatty acid moiety.The result is non-esterified carotenoids, or carotenoids no longer boundas fatty esters within the soap.

i. Natural Carotenoid-Containing Oleoresin

The natural carotenoid-containing oleoresin to be saponified is fed intothe continuous flow apparatus. Depending on how the naturalcarotenoid-containing oleoresin was extracted from the plant, suitableoleoresins may be 100% solvent free (e.g. supercritical extraction) orcontain trace amounts of solvent (for example volatile organic solventsincluding but not limited to butane and hexane). In some embodiments,the natural carotenoid-containing oleoresin is purchased from acommercial supplier. The natural carotenoid-containing oleoresin maycontain an antioxidant as it is purchased, or an antioxidant may bemixed with the natural carotenoid-containing oleoresin. In someembodiments, a surfactant, solvent, or free flow agent is mixed with thenatural carotenoid-containing oleoresin prior to the process.

ii. Antioxidant

In some embodiments, an antioxidant is additionally introduced into thecontinuous flow apparatus either as an added ingredient or as premixedwith another reagent, such as, the oleoresin. Suitable antioxidants mayinclude, but are not limited to, those listed in Section I(e) above.

The antioxidant may be provided in an amount between about 0.25% andabout 5% to the natural carotenoid-containing oleoresin on a weightbasis. In some embodiments, the antioxidant may be provided in an amountof 0.25%, about 0.5%, about 0.75%, about 1%, about 2%, about 3%, about4%, or about 5% on a weight basis to the natural carotenoid-containingoleoresin. In one preferred embodiment, the antioxidant is provided on a3% weight basis to the natural carotenoid-containing oleoresin. Theantioxidant may be provided pre-mixed with the naturalcarotenoid-containing oleoresin or may be provided separately.

iii. Proton Acceptor

Saponification occurs in the presence of a proton acceptor, or a strongbase which may be a alkaline reagent. In some embodiments,saponification is achieved in the presence of a metal hydroxide.Suitable metal hydroxides, include, but are not limited to, sodiumhydroxide, potassium hydroxide, calcium hydroxide, and magnesiumhydroxide. In one preferred embodiment, the proton acceptor is potassiumhydroxide.

The metal hydroxide is generally present in a solution which ispreferably an aqueous solution. In some embodiments, the metal hydroxidesolution ranges from about 10% to about 70%, or, in other embodimentsthe solution ranges from about 20% to about 60%. In other embodiments,the metal hydroxide is present in about a 10% solution, about a 20%solution, about a 30% solution, about a 40% solution, about a 50%solution, about a 60% solution, or about a 70% solution. In onepreferred embodiment, the metal hydroxide is a 50% aqueous solution ofpotassium hydroxide.

The ratio of natural carotenoid-containing oleoresin to metal hydroxidecan and will vary in alternate embodiments, and may be influenced by theconcentration of the metal hydroxide and the rate of introduction in thecontinuous flow apparatus. In some embodiments, the ratio of the metalhydroxide solution to the natural carotenoid-containing oleoresin isabout 10% to about 50% on a weight basis, or more preferably from about28 to about 30%. In some embodiments, the ratio of the metal hydroxidesolution to the natural carotenoid-containing oleoresin is about 10%,about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about45%, or about 50% on a weight by weight basis.

iv. Temperature

Heat is provided to the saponification step to facilitate the reaction.In general, the temperature may range from about 110° C. to about 180°C. The heat may be provided by heating devices in connection with thecontinuous flow apparatus. These devices may be set at temperaturesranging from about 110° C. to about 120° C., from about 115° C. at about125° C., from about 120° C. to about 130° C., from about 125° C. toabout 135° C., from about 130° C. to about 140° C., from about 135° C.to about 145° C., from about 140° C. to about 150° C., from about 145°C. to about 155° C., from about 150° C. to about 160° C., from about165° C. to about 175° C., from about 170° C. at about 180° C. In onepreferred embodiment, the heating is provided by a jacketing devicesurrounding the continuous flow apparatus which is set to about 140° C.

v. Pre-Heating

In one embodiment, the solution containing the proton acceptor, thenatural carotenoid-containing oleoresin, or both are pre-heated prior tocontacting. The reagents may be preheated in a reagent container priorto entering the continuous flow apparatus, or may be heated in thecontinuous flow apparatus prior to contacting of the two reagents.Heating can be provided by a variety of sources including throughconduction or convection. Exemplary heating sources include heatingjackets, heat exchangers, and the like.

Preferably, the natural carotenoid-containing oleoresin is pre-heatedand maintained in a reagent container prior to entry in the continuousflow apparatus. In such embodiments, the heated naturalcarotenoid-containing oleoresin exhibits enhanced flowability andpumpability in the continuous flow apparatus. The naturalcarotenoid-containing oleoresin may be maintained at a temperatureranging from about 50° C. to about 70° C. In certain embodiments, thenatural carotenoid-containing oleoresin is heated to a temperature ofabout 50° C., about 55° C., about 60° C., about 65° C., or about 70° C.In one preferred embodiment, the natural carotenoid-containing oleoresinis heated to a temperature of about 60° C. prior to introduction intothe continuous flow apparatus.

The proton acceptor may be pre-heated prior to contacting with thenatural carotenoid-containing oleoresin. The metal hydroxide may beheated to a temperature ranging from about 60° C. to about 150° C. Morepreferably, the metal hydroxide is heated to a temperature of aboutranging from about 80° C. to about 90° C. In alternate embodiments, themetal hydroxide is heated to a temperature of about 75° C., about 80°C., about 85° C. or about 90° C. In one preferred embodiment, the metalhydroxide is heated to a temperature of 90° C. by a plates heatexchanger within the continuous flow apparatus.

vi. Intimate Mixing

The saponification step comprises intimate mixing. Intimate mixing, asused herein, refers to high shear mixing, homogenization, such asthrough a homogenizer (including a rotor stator type homogenizer, a highpressure homogenizer), sonification, or through ultrasonification. Inone preferred embodiment, mixing is provided by a high shear mixerwithin the continuous flow apparatus. In some embodiments, intimatemixing is provided in combination with other kinds of mixing, forexample, static mixing.

(b) Atomization

The process further comprises a step where the non-esterified carotenoidsoap produced in step (a) is atomized. Methods of atomization are knownin the art. For instance, the atomization step may be achieved throughspraying the product comprising the non-esterified carotenoids producedin step (a) through a nozzle or spinning disc into the spraying chamber.A gas flow may also be introduced into the atomizing chamber as either acounter or co-current in relation to the spraying flow direction. As theliquid product from (a) is released, the soap composition containing thenon-esterified carotenoids is distributed as small droplets into the gasstream. The sprayed droplets may be dried or cooled by the gas. The gasmay be atmospheric air or an inert gas chosen from argon, nitrogen, andcombinations thereof. In some embodiments, a gas stream is used todeliver an additional free flow agent which is distributed amongst theatomized soap droplets as they pass through the atomizer.

i. Temperature

The temperature that atomization takes place at may range from about 15°C. to about 100° C. More preferably, the temperature within the atomizerranges from about 30° C. to about 80° C. In alternate embodiments, thetemperature within the atomizer is about 45° C., about 55° C., about 65°C., or about 75° C. In one preferred embodiment, the temperature in theatomizer is about 50° C.

ii. Free Flow Agent

A free flow agent may be used to enhance the flowability of the finalsoap product. The free flow agent may be introduced during atomizationthrough a gas stream in the atomizer. The free flow agent may beselected from free flow agents known in the art. Suitable examples mayinclude, but are not limited to, tricalcium phosphate, powderedcellulose, calcium stearate, magnesium stearate, sodium bicarbonate,sodium ferrocyanide, calcium ferrocyanide, potassium ferrocyanide, bonephosphate, sodium silicate, silicon dioxide, calcium silicate, magnesiumtrisilicate, talcum powder, sodium aluminum silicate, potassium aluminumsilicate, calcium aluminosilicate, bentonite, aluminum silicate, stearicacid, polydimethylsiloxane, glucose, maltodextrin, hydrophobicallymodified starch, and the like. In one preferred embodiment, the freeflow agent is silicon dioxide. In another preferred embodiment, the freeflow agent is talc.

The amount of free flow agent which is added may range from about 3% toabout 15%, or more preferably from about 5% to about 10% of the weightof the final soap product. In some embodiments, the free flow agent isabout 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about11%, about 12%, about 13%, or about 14%, or about 15% of the weight ofthe final soap product.

iii. Moisture Content

The atomizer may reduce the moisture content of the product exiting theatomizer. In certain embodiments, the moisture content of the productafter atomization is between about 5% and about 15%, more preferablybetween about 10% and about 13%. In certain embodiments, the moisturecontent of the product exiting the atomizer is about 5%, about 6%, about7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%,about 14%, or about 15%.

(c) Isomerization

The continuous flow process further comprises isomerization of thenon-esterified carotenoids, wherein the atomized soap is heated suchthat greater than 80% of the non-esterified carotenoids present are inthe all-trans isomer configuration, and the non-esterified carotenoidconcentration of the final product is greater than 10%.

Isomerization transforms cis-xanthophylls to all-trans-isomers and isgenerally achieved with thermal heating. Thermal heating may be providedto the product in a number of ways including through a fluid bed dryer,a heated vibrating conveyer, an oven or a thermal processor.

The temperature at which isomerization is conducted can and will vary indifferent embodiments, and over the course of the isomerization step.The temperature may range from about 40° C. to about 120° C. In someembodiments, the isomerization step may be conducted at a temperatureranging from about 75° C. to about 80° C., about 80° C. to about 85° C.,about 85° C. to about 90° C., about 90° C. to about 95° C., about 95° C.to about 100° C., or about 100° C. to about 105° C.

Isomerization may be conducted over a period of time from about 1 hourto about 48 hours. In some embodiments, isomerization is conducted overa period of time of about 1 hour, about 2 hours, about 3 hours, about 4hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours,about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours,about 23 hours or about 24 hours. In one embodiment, isomerizationoccurs at a temperature between about 75° C. and about 95° C. over aperiod ranging from about 1 hour to about 3 hours.

In some embodiments, isomerization may occur in more than onetemperature stage. Isomerization may be conducted at a first range oftemperatures from about 40° C. to about 60° C. Without being bound toany theory, it is thought that thermal contact at the first range oftemperatures may decrease moisture content from about 2% to about 5%.The first temperature may be maintained for a period of about 15 minutesto about 2 hours. A second range of temperatures is used to increase theall-trans isomer content. The second range of temperatures may rangefrom about 70° C. to about 90° C. and the second temperature may bemaintained for a period ranging from about 30 minutes to about 3 hours.In one preferred embodiment, isomerization is conducted at a firsttemperature of about 50° C. for about 30 minutes and at a secondtemperature of 80° C. for about 1.5 hours. In an alternate embodiment,temperature is increased over the course of the reaction from a lowertemperature ranging from about 40° C. to about 60° C. to a highertemperature ranging from about 70° C. to about 90° C.

In some embodiments, the isomerization takes place under an inertatmosphere. The inert atmosphere may be chosen, by way of non-limitingexample, from argon, nitrogen, or combinations thereof. In one preferredembodiment, isomerization is conducted in the presence of nitrogen gas.

In some embodiments, greater than 70% of the non-esterified carotenoidspresent after atomization are all-trans isomers. In other embodiments,greater than 75%, or 80%, or 85%, or 90% of the carotenoids aretransformed to the all-trans isomer. The isomerization step may increasethe total all-trans carotenoid content of the final soap product.Isomerization may increase the all-trans carotenoid content by greaterthan 5%, or greater than 10%, or greater than 15% of the final soapproduct. In one embodiment, the total all-trans carotenoid content ofthe final soap product is greater than 10%.

After isomerization the final soap product may be transferred to aproduct container. The final soap product possesses the propertiesdescribed in Section I. In some embodiments, additional agents such asfree flow agents may be added to the product after isomerization, inaddition to those added during the atomization step.

(d) Continuous Flow Apparatus

The process is conducted in a continuous flow manner in a continuousflow apparatus. By “continuous flow” it is meant that the reaction takesplace in motion within a reactor or that starting materials andconditions are continually added and withdrawn as produced. A schematicof the continuous flow apparatus and related equipment is shown inFIG. 1. While the preferred method is continuous, it is also possible toperform the method in a discontinuous fashion. That is to say, themethod may be stopped and re-started at a different time or in adifferent physical location.

Referring to FIG. 1, a first solution of the metal hydroxide within afirst reagent container 102 may be conducted through a first conduit 106to a first static mixer 112. A second solution comprising the naturalcarotenoid-containing oleoresin within a second reagent container 104may be transferred into a first static mixer 112 through a secondconduit 108. In one embodiment, reagent container 104 is heated by aheat transfer device 105. The heat transfer device may be chosen from,without limitation, electric heaters, inductive heaters, gas heaters,oil heaters, ceramic heaters and the like, or more particularly fromplates heat exchangers and tube heat exchangers. In one embodiment, aheat transfer device 110 is used to heat the metal hydroxide as itpasses through the continuous flow apparatus.

The conduits 106 and 108 may be controllable transfer pumps that pumpreagents from the reagent containers across the conduits to the firststatic mixer 112. The conduits may be controllable pressure pumps thatpressurize the reagents stored within the reagent containers topredetermined pressures selected to move the reagents out of thecontainers at desired transfer rates, and any combination thereof.Non-limiting examples of pumps suitable for use as transfer pumpsinclude gear pumps, diaphragm pumps, centrifugal pumps, piston pumps,and peristaltic pumps. In certain embodiments, the conduits areperistaltic pumps and are responsible, in addition to the size ofvarious parts of the continuous flow apparatus, for the flow rate.

The flow rate for the continuous process may vary in differentembodiments. In particular, the flow rate may be higher or lowerdepending on the production requirements and other factors known to theskilled artisan. In some embodiments, the flow rate may range between100 kg/hr and 300 kg/hr, or from 50 kg/hr to 250 kg/hr, or 50 kg/hr to300 kg/hr. In some embodiments, the flow rate may range from about 50kg/hr to about 100 kg/hr, from about 75 kg/hr to about 125 kg/hr, fromabout 100 kg/hr to about 150 kg/hr, from about 125 kg/hr to about 175kg/hr, from about 150 kg/hr to about 200 kg/hr, from about 175 kg/hr toabout 225 kg/hr, from about 200 kg/hr to about 250 kg/hr, from about 225kg/hr to about 275 kg/hr, from about 250 kg/hr to about 300 kg/hr. Inother embodiments, the flow rate may range from about 100 kg/hr to about200 kg/hr, from about 150 kg/hr to about 250 kg/hr, from about 200 kg/hrto about 300 kg/hr. In preferred embodiments, the flow rate ranges fromabout 150 kg/hr to about 250 kg/hr.

The first static mixer 112 receives reagents continuously. The firststatic mixer is generally tube-shaped and can be straight, curved, or acombination of straight and curved tubes. The static mixer contains avariety of elements inside the tubes giving patterns of flow andproviding mixing to the reagents as they pass through the tube. Theelements may provide mixing, for example, by creating laminar flow orfor example by creating radial mixing. The first static mixer has adiameter and length that, in conjunction with the flow rate, achieves aparticular residence time for the reagents.

In some embodiments, the first static mixer has a diameter of about 20mm to about 150 mm. In some embodiments, the first static mixer has adiameter from about 20 mm to about 30 mm, from about 25 mm to about 35mm, from about 30 mm to about 40 mm, from about 35 mm to about 45 mm,from about 40 mm to about 50 mm, from about 45 mm to about 55 mm, fromabout 50 mm to about 60 mm, from about 55 mm to about 65 mm, from about60 mm to about 70 mm, from about 65 mm to about 75 mm, from about 70 mmto about 80 mm, from about 75 mm to about 85 mm, from about 80 mm toabout 90 mm, from about 95 mm to about 105 mm, from about 100 mm toabout 110 mm, from about 105 mm to about 115 mm, from about 110 mm toabout 120 mm, from about 115 mm to about 125 mm, from about 120 mm toabout 130 mm, from about 125 mm to about 135 mm, from about 130 mm toabout 140 mm, from about 135 mm to about 145 mm, from about 140 mm toabout 150 mm. In one preferred embodiment, the diameter of the firststatic mixer is 50 mm.

In one embodiment, the first static mixer 112 is connected to a highshear mixer 116, which provides intimate mixing in addition to thestatic mixer. In one embodiment, a four blade slotted cylinder stator isutilized. The size of the stator can and will vary depending on otherparameters. The rotations per minute (rpm) of the blades can vary indifferent embodiments. In some embodiments, the speed ranges from 2000rpm to about 3500 rpm. In certain embodiments the speed is 2000 rpm,2100 rpm, 2200 rpm, 2300 rpm, 2400 rpm, 2500 rpm, 2600 rpm, 2700 rpm,2800 rpm, 2900 rpm, 3000 rpm, 3100 rpm, 3200 rpm, 3300 rpm, 3400 rpm, or3500 rpm.

The high shear mixer 116 may be connected directly or by way ofadditional tubing to a second static mixer 118. Where there is a secondstatic mixer, the second static mixer 118 may be tube shaped and can bestraight, curved, or a combination of straight and curved. In someembodiments, the second static mixer has a diameter of about 20 mm toabout 200 mm. In some embodiments, the first static mixer has a diameterfrom about 20 mm to about 30 mm, from about 25 mm to about 35 mm, fromabout 30 mm to about 40 mm, from about 35 mm to about 45 mm, from about40 mm to about 50 mm, from about 45 mm to about 55 mm, from about 50 mmto about 60 mm, from about 55 mm to about 65 mm, from about 60 mm toabout 70 mm, from about 65 mm to about 75 mm, from about 70 mm to about80 mm, from about 75 mm to about 85 mm, from about 80 mm to about 90 mm,from about 95 mm to about 105 mm, from about 100 mm to about 110 mm,from about 105 mm to about 115 mm, from about 110 mm to about 120 mm,from about 115 mm to about 125 mm, from about 120 mm to about 130 mm,from about 125 mm to about 135 mm, from about 130 mm to about 140 mm,from about 135 mm to about 145 mm, from about 140 mm to about 150 mm. Inone preferred embodiment, the diameter of the first static mixer is 125mm.

Temperature may also be controlled during static mixing by heat transferdevices. In some embodiments, the first and second static mixers arejacketed with a heat transfer device to maintain the passing liquid at agiven temperature.

The residence time from the introduction of the reagents to the end ofthe second static mixer may range from about 30 seconds to about 5minutes, and is dependent, in part, on temperature. Generally speaking,for higher temperatures, less residence time is required. Similarly, forlower temperatures, higher residence time is required. In someembodiments, the residence time is about 30 seconds, 45 seconds, about 1minute, about 1.5 minutes, about 2 minutes, about 2.5 minutes, about 3minutes, about 3.5 minutes, about 4 minutes, about 4.5 minutes or about5 minutes. In one embodiment, the residence time is about 95 seconds.

The second static mixer is connected either directly or throughadditional tubing to the Piston Flow Reactor 120. The piston flowreactor is a tubular reactor which may be divided into separatecompartments, each comprising a smaller tubular reactor. In someembodiments the tubular reactor comprises four tubular reactors.Temperature is controlled within the piston flow reactor. In oneembodiment, the piston flow reactor is jacketed to a temperature above100° C. In one embodiment, the piston flow reactor is jacketed to atemperature ranging from about 130° C. to about 150° C. In one preferredembodiment, the jacket temperature is about 140° C. The piston flowreactor is preferably oriented upward such that the tubular apparatusdirects the flow of reagents perpendicular to the ground longer thanthey are oriented parallel to the ground. The residence time in thePiston Flow Reactor can and will vary, preferably from about 5 minutesto about 10 minutes. In particular embodiments, the residence time inthe piston flow reactor is 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9minutes, or 10 minutes. In one preferred embodiment, the residence timeis 8 minutes.

Pressure within the continuous flow apparatus may be regulated through abarometric sensing device and one or more valves 124 found along thecontinuous flow apparatus. The valves are releasably sealed to theenvironment surrounding the continuous flow apparatus. When pressurereaches a threshold level, the valves may be opened to release gas tothe environment surrounding the continuous flow apparatus. In preferredembodiments, the valve 124 is positioned before the atomizer 122.

The atomizer 122 may comprise a spinning circular disc for theintroduction of the product from the piston flow reactor. The spinningof the disc may result in a spray of the liquid. Near the spinning disc,one or more gas blowers may be present such that the gas and sprayedliquid are in contact as they move through the atomizing chamber. Dryingand/or solidification of the liquid product containing thenon-esterified carotenoids may occur at this point. In some embodiments,the one or more gas blowers may contain a stream of free flow agent fromthe free flow agent container 128. The free flow agent may additionallybe blown into the chamber such that it comes in contact with the sprayedproduct to form a dried product containing the free flow agent as itmoves through the continuous flow apparatus.

The liquid product may be introduced to the atomizer with a feed rateranging from about 100 kg/hr to about 300 kg/hour, or from about 50kg/hr to about 250 kg/hr, or from about 150 kg/hr to 250 kg/hour. Insome embodiments, the feed rate may range from about 50 kg/hr to about100 kg/hr, from about 75 kg/hr to about 125 kg/hr, from about 100 kg/hrto about 150 kg/hr, from about 125 kg/hr to about 175 kg/hr, from about150 kg/hr to about 200 kg/hr, from about 175 kg/hr to about 225 kg/hr,from about 200 kg/hr to about 250 kg/hr, from about 225 kg/hr to about275 kg/hr, from about 250 kg/hr to about 300 kg/hr. In otherembodiments, the flow rate may range from about 100 kg/hr to about 200kg/hr, from about 150 kg/hr to about 250 kg/hr, from about 200 kg/hr toabout 300 kg/hr. In a preferred embodiment the feed rate is about 150kg/hr.

In some embodiments, the spinning disk feed to the atomizer has a speedof about 10,000 rpm to about 30,000 rpm, or more preferably from about20,000 rpm to about 25,000 rpm. In particular embodiments, the spinningdisk feed is about 20,000 rpm, about 21,000 rpm, about 22,000 rpm, about23,000 rpm, about 24,000 rpm, or about 25,000 rpm.

The residence time in the atomizer can and will vary among variousembodiments. In one embodiment, the mean residence time will range fromabout 10 and about 14 seconds. In other embodiments, the mean residencetime is about 10 seconds, about 11 seconds, about 12 seconds, about 13seconds, and about 14 seconds.

The chamber dimensions for the atomizer may range from a diameter ofabout 2000 mm to about 2500 mm. In alternate embodiments, the chamberdimensions for the atomizer may range from about 2200 mm to about 2400mm. In one preferred embodiment, the chamber dimensions for the atomizerare about 2300 mm.

The gasses introduced into the atomizer are introduced with a particularflow rate. This flow rate may range from about 2500 m³/hour to about3500 m³/hour. In some embodiments, the flow rate for the gassesintroduced into the atomizer range from about 2900 m³/hour and 3100m³/hour. In a preferred embodiment, the flow rate is about 3000 m³/hour.

The atomizer is connected either directly to or through a series oftubes to the isomerization apparatus 126. The reagents from the atomizer122 are fed into the isomerization apparatus 126. The isomerizationapparatus may be a closed tank with a series of rotating circular traysinside the tank. The reagents are fed to a top rotating tray, whereadditional rotating trays are directly below the top rotating tray.After each rotation the products are dropped onto a lower tray andleveled by stationary baffles as the tray rotates. The closed tank maybe heated to a certain temperature and may additionally have a flowinggas.

From the isomerization apparatus, the reagents may be fed into a productcontainer 130. The product container may be sized and configured toaccept product from the continuous flow apparatus. In some embodiments,the product container is connected to a free flow agent container 128,which optionally may contain the same free flow agent as the atomizer122. The free flow agent may be blown into the product container 130 viaan air stream to increase the percentage of the free flow agent.

The various components of the continuous flow apparatus may be made of avariety of suitable materials. Suitable examples of materials mayinclude, but are not limited to, metal (including stainless steel,brass), glass (including, but not limited to, borosilicate glasses), andpolymers (including, but not limited to, fluorinated poly(ethylene)(FPE), fluorinated ethylene poly(propylene) (FEP), high densitypoly(ethylene) (HDPE), poly(chlorotrifluoroethylene) (PCT), poly(etherether ketone) (PEEK), poly(tetrafluoroethylene) (PTFE), poly(vinylfluoride) (PVF), perfluoroalkoxy (PFA) polymers, and combinations orcopolymers thereof).

(e) Optional Process for Liquid Formulation

In one alternative embodiment, the process produces a liquidformulation. In such embodiments, the atomization step (b) is notperformed, but rather the product produced in step (a) is isomerized asdescribed in (c) and then is quenched in a water tank. A continuous flowreactor as described in section (d) and shown in FIG. 1 may be modifiedsuch that the atomizer is not present and the isomerized product isdirected to a water tank where the isomerized product is diluted inwater.

The ratio of isomerized product to water can and will vary. In someembodiments, the solution ranges from about a 0.25% solution to about a10% solution in water. In other embodiments the solution ranges fromabout a 0.5% solution in water to about a 5% solution in water. In onepreferred embodiment the solution is about a 1% solution in water toabout a 2% solution in water.

In one embodiment, the isomerization of the non-esterified carotenoids,wherein the diluted soap is heated such that greater than 80% of thenon-esterified carotenoids present are in the all-trans isomerconfiguration, produces a non-esterified carotenoid concentration of thefinal soap product is greater than 10%.

In still another alternative embodiment, the process produces a liquidformulation. In such embodiments, the atomization step (b) is notperformed, but rather the product produced in step (a) is quenched in awater tank and then is isomerized as described in section (c).

IV. Methods of Using a Composition of the Invention

A composition of the invention may be used to increase the carotenoidcontent of various articles, including animal products. Such methodsinclude methods of increasing pigmenting efficiency. A composition ofthe invention may also be used to improve animal health or performance.Additionally, a composition of the invention may be used to aid in thepreservation of feed compositions. Each of these methods are discussedin more detail below.

(a) Methods of Increasing the Carotenoid Content of an Article

A composition of the invention may be used to increase the carotenoidcontent of an article. For instance, a composition of the invention maybe used to increase the carotenoid content of an animal feed or ananimal supplement. In such embodiments, the method comprises combining acomposition of the invention with an animal feed or an animalsupplement. For instance, an animal feed or supplement may compriseabout 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25,20, 15, 10, or 5% of an animal feed or an animal supplement.

In some embodiments, a composition of the invention may be fed to ananimal to increase the carotenoid content of certain animal products. Insuch embodiments, a composition of the invention may be fed to theanimal by itself, or as a part of an animal feed or animal supplement.For example, poultry (or other egg laying fowl) may be fed a compositionof the invention, either alone or as part of a poultry feed orsupplement to increase the carotenoid content of egg yolk, or thecarotenoid content of broiler chicken skin, feet, or other organs.

The amount of carotenoid composition to administer to an animal toincrease the carotenoid content of an animal product may be determinedusing methods well known in the art. Generally speaking the amount mayrange from between about 1 mg xanthophyll/kg complete feed to about 100mg xanthophylls/kg complete feed. In some embodiments, the amount ofcarotenoid composition to administer may be about 1, 5, 10, 15, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or morethan 100 mg xanthophyll/kg feed. In other embodiments, the amount may beabout 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg xanthophylls/kg complete feed.

A composition of the invention may be used to increase the pigmenting ofa product, especially a food product for human consumption. For example,a composition of the invention may be used to provide pigmenting to eggyolk (for a variety of fowl), the flesh of certain animals consumed byhumans (such as broiler skin, or the flesh or meat of certainaquaculture, including fish), or other organs of animals consumed byhumans. Advantageously, an equal amount of a composition of theinvention has a greater pigmenting efficiency than other carotenoid soapcompositions. For instance, compared to other carotenoid soapcompositions, a composition of the invention may have greater than 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200% increasedpigmenting efficiency. In some embodiments, a composition may havegreater than 100, 105, 110, 115, 120, 125, or 130% increased pigmentingefficiency compared to other carotenoid soap compositions.

In one embodiment, the composition of the invention is added to apoultry diet. As will be appreciated by one of skill in the art, theamount fed per day depends on the size and desired coloration. In someembodiments, a layer diet includes about 2 ppm of the composition of theinvention, about 3 ppm, about 4 ppm, about 5 ppm, about 6 ppm, about 7ppm, or about 8 ppm. In still another embodiment, a broiler dietcontains about 20 ppm, about 25 ppm, about 30 ppm, about 35 ppm, about40 ppm, about 45 ppm, or about 50 ppm of the composition of theinvention.

(b) Methods for Improving Animal Health and Performance

Another aspect of the invention provides methods for improving animalhealth and performance by providing a composition of the invention tothe animal of interest. Those of skill in the art will appreciate thatthe amount of composition provided to a particular animal can and willvary depending upon the species, sex, and age of the animal.Furthermore, a variety of health and performance parameters may beaffected by administration of a composition of the invention.

In some embodiments, a composition of the invention may be provided topoultry, such as laying chickens, broiler chickens, turkeys, and ducks.Examples of suitable health parameters may include, but are not limitedto, body weight, body condition score, body temperature, food intake,antioxidant status, markers of oxidative stress, serum protein levels,serum mineral levels, immune system function (immune stimulation),health and diversity of gut microflora, fecal bacteria, bone and jointhealth, and so forth. Non-limiting examples of suitable performanceparameters may include weight gain, feed:gain ratio, nutrientdigestibility, feed conversion ratio, egg yield, egg quality, eggshellquality, yolk color, skin color, carcass quality, carcass yield, meatgrade, meat yield, meat protein to fat ratio, and the like.

In some embodiments, a composition of the invention may be provided todairy ruminants, such as dairy cattle, dairy sheep, and dairy goats. Ina preferred embodiment, the dairy ruminant is a dairy cow. Non-limitingexamples of suitable health parameters to be assessed may include bodyweight, body condition score, body temperature, food intake, antioxidantstatus, markers of oxidative stress, serum protein levels, serum minerallevels, immune system function, health and diversity of rumenmicroflora, fecal bacteria, and so forth. Suitable performanceparameters may include, but are not limited to, milk yield, milkefficiency, milk fat, milk protein, somatic cell counts, FCM, ECM weightgain, feed:gain ratio, nutrient digestibility, feed conversion ratio,pregnancy rate, number of offspring, weight of offspring, and so forth.

In other embodiments, a composition of the invention may be fed tonon-dairy ruminants, such as beef cattle, veal, and lambs. Examples ofsuitable health parameters may include but are not limited to bodyweight, body condition score, body temperature, food intake, antioxidantstatus, markers of oxidative stress, serum protein levels, serum minerallevels, immune system function, health and diversity of rumenmicroflora, fecal bacteria, bone and joint health, and so forth.Non-limiting examples of suitable performance parameters may includeweight gain, feed:gain ratio, nutrient digestibility, feed conversionratio, carcass quality, carcass yield, meat grade, meat yield, meatprotein to fat ratio, and the like.

In still other embodiments, a composition of the invention may beprovided to swine; that is, sows, starter piglets, grower pigs, finisherpigs, and boars. Non-limiting examples of health parameters may includebody weight, body condition score, body temperature, food intake,antioxidant status, markers of oxidative stress, serum protein levels,serum mineral levels, immune system function, health and diversity ofgut microflora, fecal bacteria, bone and joint health, and the like.Examples of suitable performance parameters may include but are notlimited to weight gain, feed:gain ratio, nutrient digestibility, feedconversion ratio, wean to estrus interval, fertility rate, number ofoffspring, weight of offspring, farrowing rate, days to weaning, carcassquality, carcass yield meat grade, meat yield, meat protein to fatratio, and the like.

In additional embodiments, a composition of the invention may beprovided to horses. Non-limiting examples of health parameters mayinclude body weight, body condition score, body temperature, foodintake, antioxidant status, markers of oxidative stress, serum proteinlevels, serum mineral levels, immune system function, health anddiversity of gut microflora, fecal bacteria, bone and joint health, andthe like. Non-limiting examples of suitable performance parameters mayinclude weight gain, feed:gain ratio, nutrient digestibility, feedconversion ratio, stride length, jump distance, speed, and the like.

In additional embodiments, a composition of the invention may beprovided to aquaculture animals, such as fish, shrimp, oysters, mussels,and the like. Examples of suitable health parameters may include but arenot limited to body weight, body condition score, food intake,antioxidant status, markers of oxidative stress, serum protein levels,serum mineral levels, immune system function, health and diversity ofgut microflora, fecal bacteria, and so forth. Non-limiting examples ofsuitable performance parameters may include weight gain, feed:gainratio, nutrient digestibility, feed conversion ratio, shell quality,carcass quality, carcass yield, meat grade, meat yield, meat protein tofat ratio, and the like.

In still further embodiments, a composition of the invention may be fedto companion animals such as cats, dogs, and the like. Examples ofsuitable health parameters may include, but are not limited to, bodyweight, body condition score, food intake, antioxidant status, markersof oxidative stress, serum protein levels, serum mineral levels, immunesystem function, body temperature, health and diversity of gutmicroflora, fecal bacteria, bone and joint health, and so forth.Non-limiting examples of suitable performance parameters may includeweight gain, feed:gain ratio, nutrient digestibility, feed conversionratio, mobility, agility, quality of life, and the like.

(c) Methods of Preserving

In other embodiments, the invention encompasses a method of preserving aproduct. In particular the method comprises adding a composition of theinvention to a product to be preserved. This can be done by directlyadding the composition of the invention to a product, or by way offeeding the composition of the invention to the source animal for aproduct. For instance, in one embodiment, a method of the inventionencompasses preserving an animal feed or supplement. The methodcomprises combining the animal feed or supplement with a composition ofthe invention. In another embodiment, the method comprises contactingthe composition of the invention with a product to be preserved.

Suitable amounts include those as detailed in Section IV(a) above.

EXAMPLES

The following examples illustrate various iterations of the invention.

Example 1 Effect on Egg Yolk Color of Layers

A trial was performed with laying hens in cages to compare thepigmenting efficiency of Xamacol 40 (“X-40”) in comparison with acomposition of the present invention—Xamacol ColorTek (“XCT”) on eggyolk color of layers.

The trial lasted 7 weeks, including 3 weeks of xanthophylls depletionfeeding the “white” basal diet, followed by 4 weeks of feeding theexperimental diets. Animals (168 Isa Brown laying hens, 48 weeks old atthe beginning of the trial) were already located in the trial room withthe same cage mates from 18 weeks of age. Experimental feeds withpigments were provided after 3 weeks of xanthophylls depletion of birdswhile feeding the “white” basal diet. Feed and water were provided forad libitum consumption. The basal diets were formulated to meet orexceed the nutrient requirements of laying hens. A single basal diet wasformulated according to the expected feed consumption. Each feedingtreatment was prepared from the addition of the corresponding amount ofproduct, in a multi-step mixing schedule, to 100 kg of feed.

There were a total of 7 feeding treatments (including the “white”Control). Feeding treatments arose from the addition to the basal dietof the experimental product at the corresponding dose (Product X-40 orXCT to provide 4, 6 or 9 mg xanthophylls/kg complete feedingstuff), anegative control with no pigment addition was also included in thedesign.

Performance variables were checked and recorded per replicate everysecond week while feeding the experimental feeds (body weight, feedconsumption, laying rate, egg weigh, incidence of broken, soft shelledor dirty eggs on a daily basis). Egg yolk color was assessed at the endof the xanthophylls depletion phase (−1d & 0d), weekly for three weeks(7d, 14d, 21d) and daily during the 4^(th) week (24d to 28d) except twodays when eggs were reserved for xanthophylls analysis (22d & 23d). Alleggs laid on each single day were taken for egg yolk color assessment orxanthophylls analysis.

Results from the present trial were good according to Isa Brownstandards (2010), with 4.7% improvements in laying rate (90.6% vs.86.6%), 1.6% lower average egg weight (63.6 g vs. 64.6 g), 6.0%improvement in feed conversion ratio (FCR) (1.884 vs. 2.005), and 3%improvement in productivity (57.6 g vs. 55.9 g). The contrast forpigment effect (X-40 vs. XCT) was significant for egg weight (2.3%differences: 62.92 g vs. 64.37 g).

The analysis of eggs from week 4 demonstrated a significant effect ofpigment and dose, with significant interactions pigment×dose (indicatingthat the slopes from the linear regressions were significantlydifferent) for the variables CIE (Commission Internationale deL'Eclairage) a*, ratio CIE a*/b* and Roche Yolk Color Fan (RYCF). Foreach dose level, pigment XCT had better values than pigment X-40 for thevariables CIE b*, ratio CIE a*/b* and RYCF. Lightness was less sensibleto differences between pigments or dose. Pigment XCT had 1.6 pointshigher yellowness value as average (39.84 vs. 38.28) and 2 points higherintercept value. For CIE a*/b* and RYCF variables, pigment XCT 100 was117% as efficient as pigment X-40. Results from non linear regressionsincluding the values for 0 ppm offered similar conclusions: pigment XCTwas 122% as efficient as pigment X-40 for the yellowness value and118-119% as efficient for the ratio redness/yellowness and RYCF.

Egg yolk weight was not significantly affected by pigment or dose, whilexanthophylls content of egg yolk was significantly affected by bothfactors, with significant or almost significant interactions whenexpressed as ppm or total content respectively. The interaction meantthat the increase in xanthophylls content of egg yolk with dose washigher for pigment XCT: pigment XCT was 137-133% as efficient as pigmentX-40. Deposition rate corrected for laying rate was significantlyaffected by pigment but not by dose (interaction was not significant):pigment XCT had 0.63 points higher deposition rate on average thanpigment X-40 (3.28% vs. 2.65%), representing that pigment XCT was 124%as efficient as pigment X-40. Pigment XCT was approximately 119% asefficient as pigment X-40 for coloration of egg yolk, 135% as efficientfor xanthophylls content of egg, and 124% as efficient for xanthophyllsdeposition rate.

Example 2 Effect on Egg Yolk Color of Layers

A trial was performed with laying hens in cages to compare thepigmenting efficiency of different yellow pigments from (X-40, XCT andCompetitor Product 3 (“C3”)) on egg yolk color of layers.

The trial lasted 9 weeks, including 5 weeks of xanthophylls depletionfeeding the “white” basal diet, followed by 4 weeks of feeding theexperimental diets. Animals (305 HyLine Brown laying hens, 23 weeks oldat the beginning of the trial) were already located in the trial roomwith the same cage mates from 18 weeks of age. Experimental feeds withpigments were provided after 5 weeks of xanthophylls depletion of birdswhile feeding the “white” basal diet. Feed and water were provided forad libitum consumption. The basal diets were formulated to meet orexceed the nutrient requirements of laying hens (Hy-Line, 2009). Asingle basal diet was formulated according to the expected feedconsumption. Each feeding treatment was prepared from the addition ofthe corresponding amount of product, in a multi-step mixing schedule, to64 or 74 kg of feed.

There were a total of 16 feeding treatments. Feeding treatments arosefrom the addition to the basal diet of the experimental products at thecorresponding dose (X-40 or XCT to provide 2.5, 5, 10, 20, 40 and 80 mgxanthophylls/kg complete feedingstuff, or C3 to provide 2.5, 5, 10 and20 mg xanthophylls/kg complete feedingstuff).

Performance variables were checked and recorded per replicate everysecond week while feeding the experimental feeds (body weight, feedconsumption, laying rate, egg weigh, incidence of broken, soft shelledor dirty eggs on a daily basis). Egg yolk color was assessed at the endof the xanthophylls depletion phase (0d), weekly for three weeks (7d,14d, 21d) and daily during the 4^(th) week (22d to 28d) except one daywhen eggs were reserved for xanthophylls analysis (25d); xanthophyllsanalysis was also performed after depletion phase (−1d). All eggs laidon each single day were taken for egg yolk color assessment orxanthophylls analysis.

No significant differences were detected among treatments, pigments ordoses, for body weight (BW) at the beginning or end of the trial, or forthe BW change. No significant effect of treatment or interactiontreatment×period were detected for any of the performance variables,except for the incidence of unsaleable eggs among treatments, but thesedifferences were not related with pigment or dose.

Egg yolk color after one week of feeding the experimental diets almostreached its final values obtained after four weeks on trial. Significantdifferences were detected at all weeks among X-40, XCT, and C3 up to 20ppm and between X-40 and XCT up to 80 ppm, for almost all variablesstudied; interactions between pigments and doses were also significantdenoting a different response among pigments at increasing doses.

Comparison of non linear regressions for all three pigments up to 10 or20 ppm gave similar results, being XCT and C3 significantly moreefficient than X-40 for yellowness (from 121% to 128%, with nodifferences among XCT and C3: 95% Confidence Limits overlapping), ratioredness/yellowness (for XCT 158% as efficient as X-40; for C3 117% to120% as efficient as X-40; being XCT more efficient than C3 because 95%Confidence Limits did not overlap) and RYCF value (for XCT 159% to 161%as efficient as X-40; for C3 117% to 120% as efficient as X-40; beingXCT more efficient than C3 because 95% Confidence Limits did notoverlap). When the whole range of doses (up to 80 ppm) was used forcomparison of X-40 and XCT, results were also similar to the onesobtained with lower inclusion levels: XCT was significantly moreefficient than X-40 for yellowness (122%), ratio redness/yellowness(165%) and RYCF value (157%); also redness presented regression valueswith biological sense, being XCT 159% more efficient than X-40.

Egg yolk xanthophylls concentration and content and xanthophyllsdeposition rate were significantly higher for XCT (i.e. averagecorrected deposition rate % up to 20 ppm was 3.02, 3.64 and 3.18 forX-40, XCT and C3 respectively; and up to 80 ppm was 2.82 and 3.46 forX-40 and XCT respectively) although significant interactions weredetected between pigments and doses for concentration, total content anddeposition (either uncorrected or corrected for laying rate) ofxanthophylls in egg yolk. Xamacol 40 at 2.5 ppm had the highestdeposition rate % as a consequence of the low level of totalxanthophylls detected in this diet compared to other pigments andconsequently a lower xanthophyll intake, and not as a consequence of ahigher concentration of TX in egg yolk. Xamacol 40 had lower depositionrates than other pigments at all doses except at 2.5 ppm. If the averagecorrected deposition rate % is calculated excluding the value of 2.5 ppmand up to 20 ppm for all pigments the results were 2.52, 3.54 and 3.17for X-40, XCT and C3 respectively (ratio 100%, 141% and 126%), and up to80 ppm for X-40 and XCT were 2.48 and 3.36 respectively (ratio 100% and135%).

Total xanthophyll concentration and content in egg yolk increased atincreasing doses of pigments in the diet, and the responses could beadjusted with linear regression. The improvement in efficiency comparedwith X-40 from the ratio of slopes of linear regressions for allpigments up to 10 ppm was 190% approximately for XCT, while for C3 theimprovement did not reach statistical significance. When doses up to 20ppm were considered, the improvement was 170% approximately for XCT and147% approximately for C3.

Considering only X-40 and XCT for the whole range of doses (up to 80ppm) the ratio of slopes indicated an improvement in efficiency on eggxanthophylls content and xanthophyll deposition rate of 125% for XCT.

Xamacol CT was more efficient than X-40. The improvement for colorvariables varied depending on the variable studied but not on the rangeof doses included in the comparison (up to 10, 20 or 80 ppm);approximate improvements were: 125% for yellowness, and 160% for ratioredness/yellowness and RYCF. The improvement for total xanthophyllsconcentration and content in egg yolk was 190% up to dose 10 ppm, 170%up to 20 ppm, and 125% up to 80 ppm. For the average correcteddeposition rate % excluding the value of 2.5 ppm the improvement was137% approximately.

Competitor product 3 was more efficient than X-40. The improvementvaried depending on the variable studied but not on the range of dosesincluded in the comparison (up to 10 or 20 ppm) for color variables;approximate improvements were: 125% for yellowness, and 120% for ratioredness/yellowness and RYCF. The improvement for total xanthophyllsconcentration and content in egg yolk was 139% but not significant up todose 10 ppm, and 147% up to 20 ppm. For the average corrected depositionrate % excluding the value of 2.5 ppm the improvement was 126%approximately.

Xamacol CT was more efficient than C3. The improvement varied dependingon the variable studied but not on the range of doses included in thecomparison (up to 10 or 20 ppm) for color variables; approximateimprovements were: 134% for ratio redness/yellowness and RYCF. Theimprovement for total xanthophylls concentration and content in egg yolkwas 137% but not significant up to dose 10 ppm, and 117% but notsignificant up to 20 ppm.

Example 3 Effect on Broiler Chicken Pigmentation

A feeding trial was carried out to compare the pigmenting efficacy oftwo different yellow pigments from marigold (X-40 and XCT) in broilerchickens pigmentation and performance until 47 days of age. A total of1,078 Ross 308 1-d old female chicks, from 1 to 47 days of age were usedand allocated at random to the experimental treatments. The experimentaldesign was completely randomized with 7 dietary treatments: T1, Basaldiet (Negative Control); T2: Basal diet+Product X-40 30 ppm; T3: Basaldiet+Product X-40 40 ppm; T4: Basal diet+Product X-40 50 ppm; T5: Basaldiet+Product XCT 30 ppm; T6: Basal diet+Product XCT 40 ppm; T7: Basaldiet+Product XCT 50 ppm. Treatments were replicated 7 times and wereoffered during Grower (22 to 35 days of age) and Finisher (36 to 47 daysof age) phases. The animals were housed in pens of 22 broilers per penat stocking density similar to that practiced commercially in the EU (30kg/m²).

Mash (Starter) and Pelleted (Grower and Finisher) feeds were fed adlibitum, and were based on wheat and soybean meal, with no added growthpromoter or veterinary antibiotics. Starter feeds were fed from 0 to 21days, grower feeds from 22 to 35 days and finisher feeds from 35 to 47days of age. Paracox vaccine was administered at 4 days by drinkingwater and coccidiostat was added to the grower diets.

Observations included growth, body weight, feed intake, feed efficiency,EPEF (European Production Efficiency Factor), general health, andpercent of mortality and culling. Also, skin pigmentation was measuredon carcasses after chilling. Moreover, oxidation of biceps femorismuscle (TBARs Technique) and intestinal histology was evaluated.

The data were analyzed as a completely randomized design by GeneralLineal Methods (GLM) of Statistical Analysis Software (SAS).Significance was declared when probability P≦0.05, and near significanttrend when 0.05<P≦0.10. Performance and skin color parameters wereanalyzed as 7 independent treatments and as a factorial design excludingthe negative treatment. Skin color parameters were also analyzed bylinear regression vs the ingested pigment using the GLM procedure of SASv. 9.0 (SAS, 2002) and by non-linear regression by fitting the data totwo different models using the NLIN procedure of SAS v. 9.0 (SAS, 2002).

The health of the animals was considered normal throughout the study,and no adverse events were noted. Performance of the animals was inaccordance with trial conditions (broilers raised in floor pens and fedmash/pelleted diets). No significant differences were observed in bodyweight of animals between treatments, between types of pigment orbetween the different dosages used at any of the ages studied. Asexpected, no significant differences in performance were observed from 0to 21 days, as all animals received a common diet. No significantdifferences were observed thereafter, between the type of pigments (X-40vs XCT) or between different dosages tested.

There were 20 deaths/culls (1.86%) between 0 and 21 days, 8 death/culls(0.74%) between 21 and 35 days, and 34 death/culls (3.15%) between 35and 47 days (Table 10). An unexpected significant effect in mortalityduring the finisher period (35 to 47 days) was detected due to the typeof pigment. No significant differences were observed in mortalitybetween dosages.

Skin pigmentation was measured on foot, breast (in two different areas;1: axilla, 2: central breast) and thigh in chilled carcasses of broilersat 47 days of age. Taking into account the lack of raw materialssupplying natural pigments in the basal diet, there was a cleardifference between negative controls and the rest of experimentaltreatments, and control animals always exhibited the paler colors.Differences between dosages were also detected, and color always gotdarker with increasing dosages of pigment, regardless the areaevaluated.

Differences between pigments were clearly detected by the RCF and theSpectrophotometer in all the evaluated areas. Broilers receiving thepigment XCT exhibited increased Roche levels in foot (7.0^(a) vs6.4^(b); P<0.0001), in breast (7.1^(a) vs 6.5^(b) and 7.9^(a) vs6.9^(b), for Point 1 and 2, respectively; P<0.0001) and thigh (6.8^(a)vs 6.2^(b); P<0.0001) than birds fed on the pigment X-40. Also, a* andb* values of breast and thigh of carcasses of pigment XCT birds werealways significantly higher than the values for pigment X-40 (1.48 vs1.14 and 28.09 vs 26.05 for a* and b* in Breast Point 1, respectively;P<0.05; 2.11 vs 1.68 and 33.41 vs 30.27 for a* and b* in Breast Point 2,respectively; P<0.05; 0.90 vs 0.75, P=0.10 and 25.89 vs 24.60, P=0.0002;for a* and b* in Thigh, respectively). The effect of level inclusion ofthe pigments in the diet was also clear and significant. Roche color fanand a* and b* values increased and L* values decreased in all the areasevaluated as the dosage of pigment augmented.

In general, linear equations differed between pigments, and the slopesof the equations for pigment XCT were higher than the slopes for pigmentX-40 for a* value in breast 2 (+25.7%; P<0.001), for b* value in breast(+13.8%; P=0.0655), breast 2 (+27.8%; P<0.0001) and thigh (+8.5%;P=0.0491) and for RCF values in foot (+7.6%; P=0.0384), breast area 2(+9.8%; P=0.0044) and thigh (+7.1%; P=0.0565). Non-linear colorsaturation functions were also evaluated, and the K values (pigmentconcentration needed to achieve half of maximum pigmentation) forpigment X-40 were higher than the K values for pigment XCT.

The effect of treatment on TBARS level in the Biceps femoris muscle ofbroilers of 47 days of age after 0 and 10 days of refrigerated storagewas also evaluated. No significant differences in lipid oxidation ofmeat samples stored 0 or 10 days at 4° C. were detected betweentreatments, between the types of pigments (X-40 vs XCT) or betweendifferent dosages tested.

The effect of treatment on villus height, crypt depth and muscular layerthickness measured on five GIT sections was also evaluated. Nosignificant differences were detected between the negative control andthe other treatments in any of the evaluated histological parameters. Ingeneral, no significant differences between pigments were detected inthe evaluated areas. There was a clear difference between negativecontrols and the rest of experimental treatments in skin pigmentation,and control animals always exhibited the paler colors in all the areasevaluated. Differences between pigments were clearly detected by the RCFand the Spectrophotometer in all the evaluated areas. Broilers receivingthe pigment XCT exhibited increased Roche levels, a* and b* values thanbirds fed on the pigment X-40. In general, linear equations differedbetween pigments, and the slopes of the equations for pigment XCT werehigher than the slopes for pigment X-40 for a* value in breast 2(+25.7%), for b* value in breast (+13.8%), breast 2 (+27.8%) and thigh(+8.5%) and for RCF values in foot (+7.6%), breast area 2 (+9.8%) andthigh (+7.1%). In general, the non-linear color saturation functionsdiffered between pigments, and the K values for pigment X-40 were higherthan the K values for pigment XCT.

Example 4 Effect on Broiler Chicken Pigmentation

A trial was performed with broiler chickens in floor pens to compare thepigmenting efficiency of different yellow pigments (X-40, XCT and C3) onbroiler coloration.

The trial lasted 6 weeks, including 3 weeks of feeding the “white” basaldiet, followed by 3 weeks of feeding the experimental diets. Animals(1223 one day old Ross 308 female broiler chickens) were randomlyallocated in 56 pens upon arrival. Experimental feeds with pigments wereprovided after 3 weeks of feeding the “white” basal diet. Feed and waterwere provided for ad libitum consumption. The basal diets wereformulated to meet or exceed the nutrient requirements of chickens(Ross, 2006). Two basal diets were formulated according birds' age(starter for 3 weeks and grower thereafter). Each feeding treatment wasprepared from the addition of the corresponding amount of product, in amulti-step mixing schedule.

There were a total of 10 feeding treatments. Feeding treatments arosefrom the addition to the basal diet of the experimental products at thecorresponding dose (X-40 or XCT provide 20, 40, 60 and 80 mgxanthophylls/kg complete feedingstuff, or C3 to provide 40 mgxanthophylls/kg complete feedingstuff).

Performance variables were checked and recorded per replicate everythird week; litter quality was subjectively evaluated at the end of thetrial. Broiler coloration was assessed at the slaughterhouse oncarcasses cooled for one day.

Neither pigments nor dose affected performance or litter quality.

Xamacol CT reached better coloration than X-40 for lightness of pectoralpterilium (60.65 vs. 61.09), yellowness of axilar apterium (28.78 vs.28.03), and lightness, redness and ratio redness/yellowness of foot pad(65.60 vs. 66.16, 5.27 vs. 4.36 and 0.11 vs. 0.09 for lightness, rednessand ratio redness/yellowness respectively). Differences in othervariables were not significant, as well as interactions pigments×dose.

Linear regressions on redness and ratio redness/yellowness of foot padpresented significant differences in intercepts, being XCT 0.9 pointshigher for redness and 0.02 points higher for ratio redness toyellowness than X-40. Improvement in efficiency measured as the ratio ofslopes (for linear regressions) or parameter k (for non linearregressions) ranged from 103% for yellowness of pectoral pterilium andfan color of tarsus, to 109% for fan color of skin, and 120% for rednessand ratio redness/yellowness of foot pad. However, none of theseimprovements reached significance.

Coloration obtained with the competitor (C3) at 40 ppm did notsignificantly differ from that obtained with X-40 or XCT at 40 ppm forany variable studied but for lightness of foot pad (lower value for C3than X-40 or XCT; 64.3^(d), 66.4^(b) and 65.8^(bcd), respectively) andyellowness of foot pad (lower value for C3 than X-40, not for XCT;43.6^(de), 47.7^(c) and 47.2^(cd), respectively).

Coefficients of variation of X-40 were higher compared to XCT forredness of pectoral pterilium and foot pad (90.3^(a) vs. 78.1^(b);62.5^(a) vs. 52.2^(b), respectively); however the CV of X-40 was lowercompared to XCT for ratio redness/yellowness of foot pad (0.09^(b) vs.0.11^(a)), but not in other variables. The lowest CV were obtained byfan colors of skin, followed by lightness and yellowness in anylocation, and fan color of foot pad. Redness and ratioredness/yellowness had much higher CV. The CV obtained in pectoralpterilium, axilar apterium, or foot pad were of similar ranges.

Example 5 Stability Studies

Tests were performed to compare the stability of a composition of theinvention containing a free flow agent against X-40. One study measuredthe reduction in total xanthophylls (“TX”) for XCT with a free flowagent in an opened bag at room temperature. Accordingly, the resultsrepresent degradation in open bag conditions (exposure to oxygen,moisture, etc). The results are shown in TABLE A. A similar study wasconducted at elevated temperatures (50° C.). The results are shown inTABLE B. TABLE C shows the degradation of TX at room temperature forboth XCT and X-40 in an oxygen permeable dark bag. TABLE D shows theresults of XCT against X-40 where both are mixed with a feed at roomtemperature in oxygen permeable dark bags. No post-manufactureencapsulation was used for XCT in any of the examples.

TABLE A Stability of XCT with and without free flow agent (FFA) at roomtemperature in an oxygen permeable bag. 1 month 3 months 6 months 9months Stability (opened TX % TX % TX % TX % bag) at room temp. (mg/g)stab. (mg/g) stab. (mg/g) stab. (mg/g) stab. XCT without FFA 88.98Quant. 83.94 94% 81.46 96% 79.36 93% XCT with FFA 74.30 Quant. 74.49Quant. 66.23 92% 68.55 95% XCT without FFA 99.15 Quant. 91.32 92% 89.6590% 85.23 86% XCT with FFA 85.51 Quant. 77.11 90% 72.86 86% 69.26 82%

TABLE B Stability of XCT with and without FFA at 50° C. in impermeablebag. 12 weeks 18 weeks 6 months 9 months Stability (closed TX % TX % TX% TX % bag) at 50° C. (mg/g) stab. (mg/g) stab. (mg/g) stab. (mg/g)stab. XCT without FFA 101.6 Quant. 97.3 98% 95.0 96% 89.2 90% XCT withFFA 83.7 99% 84.0 99% 80.4 95% 72.9 86% XCT without FFA 94.4 Quant. 93.8Quant. 85.7 Quant. 81.9 96% XCT with FFA 79.3 Quant. 69.8 97% 67.4 93%65.9 91% *quant. = no degradation is detected.

TABLE C Stability of XCT against X-40 at room temperature in an oxygenpermeable dark bag. 1 month 3 months 4 months 6 months Stability (openedTX % TX % TX % TX % bag) at room temp. (ppm) stab. (ppm) stab. (ppm)stab. (ppm) stab. Xamacol 40 26.6 62% 25.4 60% 24.0 56% 20.5 48% XCT99.0 Quant. 99.2 Quant. 91.3 92% 89.6 90% *quant. = no degradation isdetected.

TABLE D Stability of XCT against X-40 mixed with feed at roomtemperature in oxygen permeable dark bags. 1 month 3 months 4 months 6months Stability (opened TX % TX % TX % TX % bag) at room temp. (ppm)stab. (ppm) stab. (ppm) stab. (ppm) stab. X-40 mixed with Feed 382 96%248 63% 192 50% 117 30% XCT mixed with Feed 339 93% 303 83% 270 80% 31587%

Example 6 Microscopy Comparisons with Xamacol 40

Xamacol ColorTek, (XCT) as prepared as described herein was analyzed byPolarizing and Optical Microscopy. FIG. 2A shows the Xamacol 40 (X-40)soap under Polarizing Microscope at 100 magnifications. FIG. 2B showsXCT under Polarizing Microscope at 100 magnifications. The luteinnano-particles observed in X-40 soap are shown as isotropic material byPOM. FIG. 1B shows that the particle size for XCT is too small to beanalyzed. FIG. 3A. shows Xamacol 40 under an optical microscope at 400magn. FIG. 3B shows XCT under an optical microscope at 400magnifications.

Example 7 Differential Scanning Calorimetry

The composition was compared to Xamacol 40 with by Differential Scanningcalorimetry at 10° C./min in order to determine the phase transitions.Endothermic processes are shown as negative peaks or valleys andexothermic processes are shown as positive peaks. FIG. 4 shows a redcurve for X-40 and a black curve for XCT. In the case of Xamacol 40 soapappears a thin endothermic peak at 158° C. that could correspond withthe melting point of lutein crystals.

In order to determine what it is happening in each transition, bothsamples were observed under a Polarizing Microscope equipped with aheating device with the objective of visualizing the phase transitionsin soap samples at different temperatures. The first endothermic valleyfor both samples was a small melting at 58-60° C. that not producechanges in the soap. After that, a new wide endothermic peak stars (forXCT it starts at 62-64° C. and for X-40 it starts at 78-80° C.). In thisprocess the majority of the soap is melted. This transition isoverlapped with another endothermic peak (it starts more or less at 110°C.) that corresponds to the lutein nano-particles melting in bothXamacol 40 and XCT. This process finishes in both cases at 145° C. ForXCT no more transitions were observed. For Xamacol 40, a part of thelutein micro-crystals were melted before 150° C. Finally, X-40 samplewas heated until 200° C. to study the last endothermic peak. Bymicroscope the melting of lutein micro-crystals was observed more orless at 180-190° C. The last endothermic thin peak corresponds to themelting point of some lutein micro-crystals that belongs to X-40 soap.Part of the lutein micro-crystals melt before 150° C. Luteinnano-particles melt between 110-150° C. or they could be solved by themelted soap. There is an important difference between X-40 soap DSCthermogram and XCT corresponding to the micro-crystals melting at160-170° C. Wide endothermic peaks may be due to the melting ofamorphous particles and narrow endothermic peaks are due to purecrystals melting.

By DSC, the most important difference is between 70-120° C. where XCThas a large endothermic peak and X-40 has a very small one.

After studying the DSC curves, it can be concluded that thesaponification of marigold oleoresin to obtain XCT is not performedabove the lutein melting point. The main reason crystals are obtained at70-80° C. is the low soap movement coupled with enough viscosity toallow organization of the lutein molecules. But at 130-140° C., the highmovement of soap molecules doesn't allow for crystallization.Photographing the melting of X-40 shows that at 80° C. only luteincrystals (both nano and micro-particles) were observed. At 150° C. onlylutein micro-crystals were observed. At 200° C. the melting of thelutein micro-crystals was visualized. Movement at 100° C. makesobtaining a photo difficult. Photos are shown at FIGS. 5A-D.

Example 8 X-Ray Diffraction

Three samples of a composition of the invention (XCT), Xamacol 40 soapand pure lutein were analyzed by X-ray diffraction (XRD) in order todetermine the crystallinity of lutein in Xamacol 40 and XCT.

The crystalline part of X-40 presents 4 peaks at 4.44, 6.57, 6.85 and8.89 (4.44×2). Among the peaks there are two groups. The first can begrouped together as broad peaks (4.44, 6.85 and 8.89° 2θ) thatcorrespond to a crystal phase with very small size <30 nm. The sharperpeak at 6.57° 2θ is due to a crystal particle greater than 150 nm.

The crystalline part of XCT is represented by 2 peaks, 4.69 and 6.96°2θ. The peak at 4.69 is the same as the peak at 4.44 in Xamacol 40. Thispeak is due to a crystal size lower than 30 nm.

Example 9 Manufacturing Process XCT

A 50% aqueous solution of KOH was added to a reagent container in acontinuous flow apparatus. Marigold oleoresin was added to a secondreagent container in a continuous flow reactor. The marigold oleoresinwas heated to a temperature of 60° C. The KOH solution was pumped intothe continuous flow apparatus at a feed rate of about 46 kg/hour.Marigold oleoresin was pumped into the continuous flow reactor at a feedrate of about 154 kg/hour. The KOH was heated through a plates heatexchanger prior to entry into the static mixer. The temperature of theKOH was increased to about 90° C. The KOH and the marigold oleoresinwere mixed in a static mixer which was jacketed with a jackettemperature of 140° C. The residence time through the static mixer wasabout 21 seconds. Prior to entry in another static mixer, the solutionwas sent through a rotor-stator type homogenizer. After the rotor-statorhomogenizer, the solution was passed through a second static mixer whichwas jacketed with a jacket temperature of 140° C. The residence time inthe second static mixer was about 72 seconds. The solution was thenpassed through a piston flow reactor with a jacket temperature of 140°C. The residence time in the piston flow reactor was about 8 minutes.Following the piston flow reactor, the solution was passed through anatomizer, followed by a dryer. SiO₂ was blown onto the atomized product.The product is not encapsulated.

Example 10 Comparative Manufacture for X-40

Marigold oleoreisn, E-484 (glyceryl polyethyleneglycol ricinoleate) andpropylene glycol were mixed together inside a saponification reactor.The temperature in the saponification reactor ranged from 58° C. toabout 63° C. The time lapse for this step was about 10′. Aqueous sodiumhydroxide was then poured into the saponification reactor. The reactionbetween the marigold oleoresin and the potassium hydroxide was allowedto occur for 2 to 3 hours. The temperature during this time was 75-80°C. Phosphoric acid was then added to the saponification reactor toquench excess potassium hydroxide. The temperature rose from 5 to 10° C.because of the exothermic nature of this reaction. The soap was pouredinto a paddle mixer loaded with calcium carbonate and silicon dioxide.The soap was adsorbed by the free flow agent. The total time for this isabout 20′ including loading and mixing. The mixer was discharged andpassed through a grinder in order to disaggregate lumps. The product issieved through a vibrating screen. A schematic is shown in FIG. 6. TABLEE Shows physical properties of the final product prepared by thisprocess and by the process of the invention. The product of this processis referred to as X-40 throughout the application.

TABLE E Comparison of physical properties between XCT and X-40 PhysicalProperty XCT X-40 Bulk aspect Yellow-brown powder Orange-yellow finepowder Xanthophyll crystal size 99% below 0.5 microns 64% below 0.5microns Particle aspect Spheroid to elongated particles HeterogeneousParticle distribution 100% < 0.85 mm 95% < 0.85 mm 98% > 0.105 mm Thereisn't a specification about the maximum content of very fine particlesParticle strength withstands pressure up to withstands pressure up to100 g/cm² 100 g/cm² Melting point Above 90° C. Does not melt completelySolid excipients Talc/SiO₂ CaCO₃/SiO₂ Maximum solid excipients Max 12%67% content Dustiness 84 50.7 Bulk density (g/ml) 0.44-0.53 0.6-0.8 Max.Moisture (%)  2 8  Xanthophyll content (mg/g) 100.0-104.0 40-42 Min. alltrans xanthophylls (%)   82.0 88.0

Example 11 Stability Study with Ethoxyquin

Compositions of the invention (XCT) were studied with different amountsof ethoxyquin (ETQ). The samples were prepared as described in Example9. The samples were measured by using a validated analytic method todetermine total xanthophylls. Total xanthophylls were analyzed for eachsample at the beginning of the study. All samples were stored in anoxygen permeable dark plastic bag at 25° C. All samples were held in thesame conditions (darkness, oxygen, temperature, light, and time).

As can be seen in TABLE F, there were no difference between productswith different amounts of ethoxyquin. Instead, the compositionsmanufactured in accordance with the present invention were more stableand had more effect than that produced by the addition of ethoxyquin.

TABLE F Stability Study with Ethoxyquin XCT stability 15 days 1 month 3months 6 months 9 months with different % % % % % ETQ content Initial TXstab TX stab. TX stab. TX stab. TX stab. XCT (3% ETQ) 91.96 90.1 98%88.5 96% 81.2 88% 78.6 85% 75.8 82% XCT (3% ETQ) 83.39 81.6 98% 80.1 96%74.3 89% 74.1 89% 73.7 88% XCT (0.7% ETQ) 89.37 83.0 93% 84.0 94% 80.890% 78.9 88% 78.0 87% XCT (ETQ free) 102.35 94.2 92% 91.0 89% 91.6 90%91.0 89% 91.6 89% XCT (ETQ free) 92.7  0% 89.8 97% 81.2 88% 75.6 82%73.8 80%

Example 12 Comparative Stability Study

Several comparative studies were conducted under different temperatureconditions, with pure composition or mixed with a vitamin mineralpremix. Stability studies were conducted as described in Example 11. Thecompositions were tested against compositions prepared as described inExample 10 (X-40), competitor 1, and competitor 2. Competitor 2 was sodegraded that measurements taken after 30 days were discontinued. Theresults under different conditions and as mixed with a vitamin premixare shown in TABLES G-I.

TABLE G Stability Study - Room Temperature, Open Bag Room 15 days 30days Temperature TX % % Open Bag Initial Initial TX stab. TX stab. XCT100.1 99.0 103.0 Quant. 95.9 98% 98.4 100.7 97.9 98.5 101.5 98.5 X-4042.5 42.3 34.1 80% 29.2 70% 42.4 34.0 29.8 41.9 33.8 30.0 Competitor 122.5 23.2 21.5 97% 20.8 88% 24.5 23.2 19.1 21.8 23.0 21.5 Competitor 218.7 19.1 13.1 65% 7.4 40% 19.5 11.9 7.5 19 12.1 7.8 45 days 60 days 90days 4 months 6 months Room Temp. % % % % % Open Bag TX stab. TX stab.TX stab. TX stab. TX stab. XCT 97.6 Quant 95.6 98% 97.7 97% 97.5 98%94.0 95% 99.4 96.1 96.7 96.8 94.4 99.9 98.8 93.8 95.4 94.3 X-40 29.4 71%29.4 70% 29.3 70% 29.3 68% 29.0 69% 30.0 29.7 29.7 28.2 29.0 30.4 30.229.6 28.3 29.5 Competitor 1 22.2 92% 17.1 77% 19.0 78% 17.6 79% 17.5 78%20.9 17.9 19.2 19.3 17.2 20.7 18.3 16.4 18.1 19.4 Competitor 2Discontinued

TABLE H Comparative Stability Study, 45° C., Open Bag 15 days 30 days 45days 60 days 90 days 45° C. TX % % % % % open bag Initial Initial TXstab. TX stab. TX stab. TX stab. TX stab. XCT 100.1 99.0 101.4 Quant100.0 98% 92.2 94% 89.7 92% 86.5 87% 98.4 104.2 96.1 93.0 91.7 86.7 98.598.4 96.2 94.8 92.6 84.1 X-40 42.5 42.3 31.4 75% 29.1 69% 27.2 65% 24.859% 19.3 46% 42.4 31.8 29.1 27.4 25.2 20.2 41.9 32.5 29.6 27.8 24.3 19.5Competitor 1 22.5 23.2 19.0 82% 15.4 65% 10.9 44% 9.0 39% 9.6 42% 24.519.1 14.6 9.6 8.8 10.0 21.8 18.7 15.5 10.2 9.3 9.8 Competitor 2 18.719.1 4.2 21% discontinued 19.5 3.9 19 4.0

TABLE I Comparative Stability Study, 45° C., Open Bag with a Vitamin RTopen bag, 15 days 30 days Vitamin Mineral TX % % Premix Initial InitialTX stab. TX stab. XCT + Vitamin 2.1 2.08 2.0 96% 1.9 92% Mineral Premix2.1 2.0 1.9 2.1 2.0 1.9 X-40 + Vitamin 2.0 1.91 1.4 70% 1.3 64% MineralPremix 1.8 1.3 1.2 1.9 1.3 1.2 Competitor 1 + 3.7 3.74 3.7 99% 4.0Quant. Vitamin 3.6 3.5 4.1 Mineral Premix 3.9 3.9 3.9 Competitor 2 + 2.02.01 1.1 59% 0.9 44% Vitamin 2.1 1.2 0.9 Mineral Premix 2.0 1.2 0.9 RTopen bag, 45 days 60 days 90 days 4 months 6 months Vitamin Mineral % %% % % Premix TX stab. TX stab. TX stab. TX stab. TX stab. XCT + Vitamin1.8 92% 1.7 86% 1.7 80% 1.8 83% 1.5 75% Mineral Premix 1.9 1.8 1.7 1.71.5 1.9 1.8 1.6 1.7 1.6 X-40 + Vitamin Discontinued Mineral PremixCompetitor 1 + 3.3 90% 3.0 77% 2.4 68% 2.6 74% 2.5 67% Vitamin Mineral3.5 2.6 2.7 2.7 2.5 Premix 3.4 3.2 2.5 2.9 2.6 Competitor 2 +Discontinued Vitamin Mineral Premix *quant = no degradation is detected.

Example 13 Closed Bag Stability Studies

Total xanthophylls were studied in closed bag conditions to simulatepacking. All samples were manufactured as described in Example 9. Thesamples (with or without a free flow agent) were stored in impermeablealuminum sealed plastic bag at 25° C. All samples were run under thesame conditions of low oxygen content, temperature, and darkness. Allformulations showed good stability at 24 months, as shown in TABLE J.The samples had approximately 18% silicon dioxide as a free flow agent(FFA).

TABLE J Stability of XCT and Silicon Dioxide Stability Initial 15 days 1month 2 months 3 months Closed bag with TX TX % TX % TX % TX % lowoxygen (mg/g) (mg/g) stab. (mg/g) stab. (mg/g) stab. (mg/g) stab. XCTwithout FFA 85.05 85.5 Quant. 89.0 Quant. 81.8 96% 83.9 94% XCT with FFA72.29 73.6 Quant. 74.3 Quant. 69.9 97% 74.5 Quant. XCT without FFA 99.4599.0 Quant. 99.1 Quant. 99.2 Quant. 91.3 92% XCT with FFA 84.53 84.6Quant. 85.5 Quant. 79.4 94% 77.1 90% Stability (continued) 6 months 9months 12 months 18 months 24 months Closed bag with TX % TX % TX % TX %TX % low oxygen (mg/g) stab. (mg/g) stab. (mg/g) stab. (mg/g) stab.(mg/g) stab. XCT without FFA 81.5 96% 79.4 93% 79.5 93% 79.8 94% 74.588% XCT with FFA 66.2 92% 68.6 95% 69.1 96% 65.4 90% 63.7 88% XCTwithout FFA 89.6 90% 85.2 86% 79.5 80% 83.8 84% 78.8 79% XCT with FFA72.9 86% 69.3 82% 65.7 78% 66.4 79% 65.8 78%

Example 14 Powder Formulation

The below represents an Exemplary powder formulation for the XCT processexpressed as percent weight.

TABLE K Exemplary Powder Formulation XCT Composition marigold soap75%-95% SiO₂ 0%-5% Talc 2%-10% Stearic acid 0%-7% moisture 0.3%-1% ETQ0.6%

Example 15 Ethoxyquin-Free Formulation

The below represents and Examplary powder formulation without ethoxyquinfor the XCT process.

TABLE L Exemplary Powder Formulation without Antioxidant XCT powder ETQfree Composition marigold soap 76%-95% SiO₂ 0%-5% Talc 2%-10% Stearicacid 0%-8% KOH 0.1%-1% moisture 0.3%-1%

Example 16 Liquid Formulation

Liquid formulations are made in a similar fashion to powderformulations. However, instead of the marigold soap being sprayed intoan atomizer, it was sprayed into water. The water was maintained at15-30° C. With the addition of soap the temperature was increased to40-50° C. The mixture was mixed over the course of one hour with anultraturrax. The liquid product was heated at 70-80° C. for 6-9 hours toachieve isomerization to the desired levels of trans lutein andtrans-zeaxanthin. After cooling the liquid product is finished.

TABLE M Exemplary Liquid Formulation XCT liquid Composition marigoldsoap 12%-17% ETQ 0.6% E-484 1% Water 82%-87%

Example 17 Stability Studies with Liquid Formulation

The liquid formulation is a stable emulsion of marigold soap in water.The stability of a liquid formulation, the stability is mainly due tolow contact with oxygen. The results are shown in TABLE N.

TABLE N Stability Study with Liquid Formulations Initial 3 months 6months 12 months TX TX % TX % TX % Stability at RT (mg/g) (mg/g) stab.(mg/g) stab. (mg/g) stab. Xamacol 15 15.7 — — 16.4 Quant. 16.3 Quant.(X-40 in liquid) XCT 12/006 16 16.4 Quant. 15.4 96% 16.3 Quant. liquidpH 12.5 XCT 12/006 15.9 15.9 Quant. 17.5 Quant. 15.8 99% liquid pH 13*quant = no degradation is detected.

Example 18 Comparative Studies Against Products

Stability studies were conducted as described in Examples 5 and 11 andcompared to apo-ester products. The below table provides the results ofthese studies against Competitor 4 (C4) and Competitor 5 (C5). For theapo-ester column. Where two values are present, the first is C4 and thesecond is C5. FIG. 7A shows the results against C4 and C5. FIG. 7B showsthe results against C3 and C6.

TABLE O Comparitive Study With Apo Ester Time Apo-ester Storageconditions (months) XCT % X40% %* Open bag 25° C. 1 98 75 100-100 3 9467  95-100 6 91 30 98-88 9 88 —  87 (C5) 12 87 —  85 (C4) Open bag - 25°C. 6 92.5 — 90-98 ETQ low Open bag - 25° C. 6 87.5 — — ETQ free Open bag50° C. 6 94 67 90-95 Open bag 75° C. 48 h 100 55 100 (C4) In vitamin 25°C. 3 76 — 81-96 premix - 25° C. 3 69 15 — open bag In feed - 25° C. 397.5 60-65 100-100 open bag 45° C. 1 100 — 100-100 Closed bag 25° C. 12100 100 100 (C4)

Example 19 Yellow Pigments of Egg Yolk Color of Layers

A trial was performed with laying hens in cages to compare thepigmenting efficiency of different yellow pigments (A, B and C) on eggyolk color of layers. The trial lasted 9 weeks, including 5 weeks ofxanthophylls depletion feeding the “white” basal diet, followed by 4weeks of feeding the experimental diets.

Animals: 305 HyLine Brown laying hens, 23 weeks old at the beginning ofthe trial.

Feed and water were provided for ad libitum consumption. The basal dietswere formulated to meet or exceed the nutrient requirements of layinghens (Hy-Line, 2009). Do we need more info for this cite? A single basaldiet was formulated according to the expected feed consumption. Eachfeeding treatment was prepared from the addition of the correspondingamount of product.

There were a total of 16 feeding treatments (6 replicates of 3 hens/cageper treatment). Feeding treatments arose from the addition to the basaldiet of the experimental products at the corresponding dose (Product Aor B to provide 2.5, 5, 10, 20, 40 and 80 mg xanthophylls/kg completefeedingstuff, or Product C to provide 2.5, 5, 10 and 20 mgxanthophylls/kg complete feedingstuff).

Performance variables were checked and recorded per replicate everysecond week while feeding the experimental feeds (body weight, feedconsumption, laying rate, egg weigh, incidence of broken, soft shelledor dirty eggs on a daily basis). Egg yolk color was assessed at the endof the xanthophylls depletion phase (0d), weekly for three weeks (7d,14d, 21d) and daily during the 4th week (22d to 28d) except one day wheneggs were reserved for xanthophylls analysis (25d); xanthophyllsanalysis was also performed after depletion phase (−1d). All eggs laidon each single day were taken for egg yolk color assessment orxanthophylls analysis.

Assessment of the color was done using a Minolta colorimeter. Thecolorimeter describes color in the CIE L*a*b* L* indicates the lightnessrepresenting dark to light on a scale of (0-100). The a* (redness) valuegives the degree of the red-green color with a higher positive a* valueindicating more red color. The b* (yellowness) value indicates thedegree of yellow-blue color with a higher positive b* value indicatingmore yellow color. Results are shown in FIGS. 8A-8D. Linear regressionon the variables for all pigment doses up to 20 ppm, and during thefourth week of the experimental diets showed that XCT was 121% asefficient as X-40 for yellowness. XCT was 159% as efficient as X-40 forthe RYCF.

Total xanthophyll content of the egg yolks (taken after what time) isshown in FIGS. 9A and 9B. Linear regression analysis shows that at day25, XCT was 172% as efficient as X-40 for egg xanthophyllsconcentration.

Example 20 Layer Trials with No Red Pigment Added

A trial was performed with laying hens in cages to determine thecomparative efficacy of Xamacol ColorTek vs. apoester (C4) in impartingyellow color to egg yolk.

The trial lasted 9 weeks, including 5 weeks of xanthophylls depletionfeeding the “white” basal diet, followed by 4 weeks of feeding theexperimental diets.

Animals: 216 Hy-line W-36 laying hens, 57 weeks old at the beginning ofthe trial.

This study was conducted at MRP battery facility. The house wasenvironmentally controlled. Feed and water were provided for ad libitumconsumption. All rations were designed to meet or exceed all dietaryrecommendations for W-36 (based on Hy-line guide 2009-11).

There were a total of 9 treatments with 24 pen (cage) replicates pertreatment. Each cage had one hen.

Performance variables were checked and recorded per replicate everysecond week while feeding the experimental feeds (body weight, feedconsumption, laying rate, egg weigh, incidence of broken, soft shelledor dirty eggs on a daily basis). Egg yolk color was assessed at the endof the xanthophylls depletion phase (0d), weekly for three weeks (7d,14d, 21d) and more frequently during the 4th week (25d to 28d). All eggslaid on each single day were taken for egg yolk color assessment. TABLEP shows the treatments.

TABLE P Treatments Treatments Description 1 T1: “0” pigmentsupplementation (control) 2 T2: 2 ppm XCT 3 T3: 4 ppm XCT 4 T4: 6 ppmXCT 5 T5: 9 ppm XCT 6 T6: 12 ppm XCT 7 T7: 2 ppm of Apo-ester (C4) 8 T8:4 ppm Apo-ester (C4) 9 T9: 6 ppm Apo-ester (C4)

Results are shown in FIGS. 10A-10D. Linear regression on the variablesfor all pigment doses up to 80 ppm, and during the fourth week of theexperimental diets showed that XCT was 159% as efficient for rednessvalue than X-40, that XCT was 122% for yellowness value as X-40, XCT was165% as efficient as X-40 for the ratio of redness/yellowness, that XCTwas 157% as efficient as X-40 for RYCF.

Total xanthophyll content is shown in FIGS. 11A-11B. Linear regressionof egg xanthophyll content and xanthophyll deposition rate showed thatXCT was 126% as efficient as X-40 of egg xanthophyll concentration, andXCT was 124% as efficient as X-40 for egg xanthophyll content. XCT was131% more efficient than competitor product 3 in redness/yellownessratio, 133% more efficient than competitor product 3 for RYCF, and 117%more efficient than competitor product 3 for total xanthophylls in eggyolk.

Similar comparisons were made with Ester products. Results are shown inFIGS. 12A-12D.

Example 21 Layer Trials with Synthetic Red Pigment Added

A trial was performed with laying hens in cages to compare thepigmenting efficiency of 2 yellow pigments (A and B) when red pigmentfrom synthetic origin (C) was added on egg yolk color of layers.

The trial lasted 7 weeks, including 3 weeks of xanthophylls depletionfeeding the “white” basal diet, followed by 4 weeks of feeding theexperimental diets.

Animals: 360 Hy-line (Isa brown plus), 32 wk old at the beginning of thetrial.

Feed and water were provided for ad libitum consumption. The basal dietswere formulated to meet or exceed the nutrient Hy-line 2009 requirementsof laying hens (Isa Brown, 2009-10). A single basal diet was formulatedaccording to the expected feed consumption. Each feeding treatment wasprepared from the addition of the corresponding amount of product.

There were a total of 10 feeding treatments (6 replicates of 6 hens/cageper treatment).

Performance variables were checked and recorded per replicate everysecond week while feeding the experimental feeds (body weight, feedconsumption, laying rate, egg weigh, incidence of broken, soft shelledor dirty eggs on a daily basis). Egg yolk color was assessed at the endof the xanthophylls depletion phase (0d), weekly for three weeks (7d,14d, 21d) and more frequently during the 4th week (25d to 28d). All eggslaid on each single day were taken for egg yolk color assessment.

Color Fan analysis was conducted with DSM Yolk Color Fan (also known asRoche Color Fan). The analysis has 15 scales of color index used todistinguish yolk color density. The method consists of assessing yolk asa visual comparison and estimation of carotenoids. Result of the yolkcolor fan show that when red pigment is added, the CXT is comparable tomultiple competitors. FIG. 13 shows the result of color fan analysiswith red pigment and without red pigment. The differences are notdetectable to consumers.

Example 22 Layer Trial with Natural Red Pigment Added

A trial was performed with laying hens in cages to compare thepigmenting efficiency of 2 yellow pigments (A and B) when red pigmentfrom natural origin (C) was added on egg yolk color of layers.

The trial lasted 7 weeks, including 3 weeks of xanthophylls depletionfeeding the “white” basal diet, followed by 4 weeks of feeding theexperimental diets.

Animals: 240 commercial brown laying hens were used (Hy-line Brown) at38 wks old at the beginning of the trial.

Feed and water were provided for ad libitum consumption. The basal dietswere formulated according to Hy-line Brown recommendation (Hy-LineInternational Red Book, 2009). A single basal diet was formulatedaccording to the expected feed consumption. Each feeding treatment wasprepared from the addition of the corresponding amount of product.

There were a total of 10 feeding treatments (24 replicates of 1 hen/cageper treatment).

Performance variables were checked and recorded per replicate everysecond week while feeding the experimental feeds (body weight, feedconsumption, laying rate, egg weigh, incidence of broken, soft shelledor dirty eggs on a daily basis). Egg yolk color was assessed at the endof the xanthophylls depletion phase (0d), weekly for three weeks (7d,14d, 21d) and more frequently during the 4th week (25d to 28d). All eggslaid on each single day were taken for egg yolk color assessment.

Performance variables were checked and recorded per replicate everysecond week while feeding the experimental feeds (body weight, feedconsumption, laying rate, egg weigh, incidence of broken, soft shelledor dirty eggs on a daily basis). Egg yolk color was assessed at the endof the xanthophylls depletion phase (0d), weekly for three weeks (7d,14d, 21d) and more frequently during the 4th week (25d to 28d). All eggslaid on each single day were taken for egg yolk color assessment.

Color Fan analysis was conducted with DSM/Roche Yolk Color Fan (alsoknown as Roche Color Fan). The analysis has 15 scales of color indexused to distinguish yolk color density. The method consists of assessingyolk as a visual comparison and estimation of carotenoids. Result of theyolk color fan show that when red pigment is added, the CXT iscomparable to multiple competitors. FIG. 14 shows the result of colorfan analysis with red pigment and without red pigment. The differencesare not detectable to consumers.

Example 23 Spectroscopic Studies

Spectroscopic studies were performed with X-40 and XCT. FIG. 15A shows aside by side of the FTIR spectra. X-40 and XCT have similarcharacteristics with bands corresponding to vibration modes of aromaticand aliphatic functional groups. The ═C—H stretches (3033 and 3010cm⁻¹), C—H out of the ring plane (710 cm⁻¹) and C—C stretches in thearomatic ring (1560, 1473 cm⁻¹) indicate the presence of unsaturatedmoieties in the structure. In addition the bands corresponding to —C—Hstretches of the alkyl groups (2915/2849 cm⁻¹) are present in bothsamples indicating the existence of aliphatic groups. In the case of T40FTIR spectrum, the intense bands at 1400, 1063 and 1024 cm⁻¹,corresponding to phosphate groups, are overlapped with some bands in thefingerprint region of molecule. FIG. 15B shows an expanded region fromregion from 1800 to 600 cm⁻¹ The bottom and top lines are the same asFIG. 15A.

FIG. 15C shows the Raman spectra of X-40 (black) against XCT (red).

Example 24 Thin Layer Chromatography

Samples of XCT and X-40 were dissolved in chloroform and centrifuged fordiscarding the white precipitate. Thin Layer Chromatography (TLC) wascarried out in a mobile phase of hexane:ethyl acetate (1:1 v/v). TLCplates were developed by irradiating with UV and visualized using I₂exposure. The initial TLC, shown in FIG. 16A showed three principalcompounds. Silica gel chromatography columns were used to separatecompounds from the mixture samples. The initial fractions were elutedusing 2:1 hexane:ethyl acetate as a solvent (fractions 1-5). Thepolarity of the solvent mixture was then increased to ethyl acetate(fractions 6 to 14), and finally 20% methanol in ethyl acetate(fractions 15 to 19). FIG. 16B shows the results for X-40.

From the TLC is possible to conclude that the proportions of principalcompounds are not the same for X-40 and XCT. The principal compound isnumber 3 for both samples, as also was corroborated for the amountisolated in pure form by column chromatography of X-40 and XCT. In thecase of XCT compound 2 was not successfully purified from column, as isshown in FIG. 16C, while others are present in fraction 2. For XCT theamount of compound 2 is significantly less than in X-40. In addition,the amount of compound 3 purified from column was higher for XCT thanfor X-40.

What is claimed is:
 1. A process for creating a final product with anon-esterified carotenoid concentration of greater than 10%, the processcomprising: a) alkaline saponification of a naturalcarotenoid-containing oleoresin, wherein the saponification occurs inthe presence of a metal hydroxide, with intimate mixing, and occurs at atemperature between about 110° C. to about 180° C., resulting in acomposition comprising non-esterified carotenoids and oleoresincomponents from the original natural carotenoid-containing oleoresin, b)atomization of the resulting composition comprising non-esterifiedcarotenoids and oleoresin components from the original naturalcarotenoid-containing oleoresin to produce an atomized composition, andc) isomerization of the non-esterified carotenoids, wherein the atomizedcomposition is heated such that greater than 80% of the non-esterifiedcarotenoids present are in the all-trans isomer configuration, and thenon-esterified carotenoid concentration of the final product is greaterthan 10%.
 2. The process of claim 1, wherein the naturalcarotenoid-containing oleoresin is marigold oleoresin.
 3. The process ofclaim 1, wherein the natural carotenoid-containing oleoresin isprocessed to make a natural carotenoid-containing oil prior to step (a)and the resulting natural carotenoid containing oil is used in step (a).4. The process of claim 1, wherein the metal hydroxide is selected fromthe group consisting of KOH, NaOH and Ca(OH)₂.
 5. The process of claim1, wherein the natural carotenoid-containing oleoresin is heated priorto mixing with the metal hydroxide.
 6. The process of claim 5, whereinthe natural carotenoid-containing oleoresin is heated to a temperatureranging from about 50° C. to about 70° C. prior to mixing with the metalhydroxide.
 7. The process of claim 1, wherein continuous flow occurs ata rate of about 100 to about 300 kg/hour.
 8. The process of claim 7,wherein continuous flow occurs at a rate of about 150 to about 250kg/hour.
 9. The process of claim 1, wherein step (b) occurs in thepresence of between about 3 and 15% of a free flowing agent.
 10. Theprocess of claim 8, wherein step (b) occurs in the presence of betweenabout 5 and 10% of silicon dioxide.
 11. The process of claim 1, whereinmoisture content after atomization is between about 10% and 13%.
 12. Theprocess of claim 1, wherein the isomerization occurs under an inertatmosphere.
 13. The process of claim 1, wherein the isomerization occursat a temperature between about 75° C. and about 95° C. over a periodranging from about 1 hour to about 3 hours.
 14. The process of claim 1,wherein the particles of the final product are each less than about 0.9mm.
 15. A carotenoid formulation, the formulation resulting from aprocess for creating a final product with a non-esterified carotenoidconcentration of greater than 10%, the process comprising: a) alkalinesaponification of a natural carotenoid-containing oleoresin, wherein thesaponification occurs in the presence of a metal hydroxide, withintimate mixing, and occurs at a temperature between about 110° C. toabout 180° C., resulting in a composition comprising non-esterifiedcarotenoids and oleoresin components from the original naturalcarotenoid-containing oleoresin, b) atomization of the resultingcomposition comprising non-esterified carotenoids and oleoresincomponents from the original natural carotenoid-containing oleoresin toproduce an atomized composition, and c) isomerization of thenon-esterified carotenoids, wherein the atomized composition is heatedsuch that greater than 80% of the non-esterified carotenoids present arein the all-trans isomer configuration, and the non-esterified carotenoidconcentration of the final product is greater than 10%.
 16. Theformulation of claim 15, wherein the natural carotenoid-containingoleoresin is processed to make a natural carotenoid-containing oil priorto step (a) and the resulting natural carotenoid containing oil is usedin step (a).
 17. A process for creating an aqueous product from waterand a final product with a non-esterified carotenoid concentration ofgreater than 10%, the process comprising: a) alkaline saponification ofa natural carotenoid-containing oleoresin, wherein the saponificationoccurs in the presence of a metal hydroxide, with intimate mixing, andoccurs at a temperature between about 110° C. to about 180° C.,resulting in a composition comprising non-esterified carotenoids andoleoresin components from the original natural carotenoid-containingoleoresin, b) isomerization of the non-esterified carotenoids, whereinthe composition is heated such that greater than 80% of thenon-esterified carotenoids present are in the all-trans isomerconfiguration, to produce a non-esterified carotenoid concentration ofthe final product is greater than 10%, and c) contacting the finalproduct with water in sufficient amounts to create an aqueous producthaving from 0.5% to about 10% of the final product.
 18. The process ofclaim 17, wherein the natural carotenoid-containing oleoresin ismarigold oleoresin.
 19. The process of claim 17, wherein the naturalcarotenoid-containing oleoresin is processed to make a naturalcarotenoid-containing oil prior to step (a) and the resulting naturalcarotenoid containing oil is used in step (a).
 20. The process of claim17, wherein the metal hydroxide is selected from the group consisting ofKOH, NaOH and Ca(OH)₂.
 21. The process of claim 17, wherein the naturalcarotenoid-containing oleoresin is heated prior to mixing with the metalhydroxide.
 22. The process of claim 17, wherein the naturalcarotenoid-containing oleoresin is heated to a temperature ranging fromabout 50° C. to about 70° C. prior to mixing with the metal hydroxide.23. The process of claim 17, wherein continuous flow occurs at a rate ofabout 50 to about 300 kg/hour.
 24. The process of claim 23, whereincontinuous flow occurs at a rate of about 150 to about 250 kg/hour. 25.The process of claim 17, wherein the particles of the final product areeach less than about 0.9 mm.
 26. A carotenoid formulation, theformulation resulting from a process for creating an aqueous productfrom water and a final product with a non-esterified carotenoidconcentration of greater than 1%, the process comprising: a) alkalinesaponification of a natural carotenoid-containing oleoresin, wherein thesaponification occurs in the presence of a metal hydroxide, withintimate mixing, and occurs at a temperature between about 110° C. toabout 180° C., resulting in a composition comprising non-esterifiedcarotenoids and oleoresin components from the original naturalcarotenoid-containing oleoresin, b) isomerization of the non-esterifiedcarotenoids, wherein the composition is heated such that greater than80% of the non-esterified carotenoids present are in the all-transisomer configuration, to produce a non-esterified carotenoidconcentration of the final product is greater than 10%, and c)contacting the final product with water in sufficient amounts to createan aqueous product having from 1% to about 2% of the final product. 27.The process of claim 26, wherein the natural carotenoid-containingoleoresin is marigold oleoresin.
 28. The process of claim 26, whereinthe natural carotenoid-containing oleoresin is processed to make anatural carotenoid-containing oil prior to step (a) and the resultingnatural carotenoid containing oil is used in step (a).